chemical reagents and derivatization procedures in drug

Chemical Reagents and Derivatization Procedures in DrugAnalysis

Neil D. Danielson, Patricia A. Gallagher, and James J. Bao

inEncyclopedia of Analytical Chemistry

R.A. Meyers (Ed.)pp. 7042–7076

John Wiley & Sons Ltd, Chichester, 2000

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CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS 1

Chemical Reagents andDerivatization Proceduresin Drug Analysis

Neil D. Danielson and Patricia A. GallagherMiami University, Oxford, USA

James J. BaoProcter and Gamble Pharmaceuticals, Mason,USA

1 Introduction 1

2 Gas Chromatography 22.1 Alkylation 22.2 Acylation 32.3 Silylation 52.4 Sample Handling 7

3 High-performance LiquidChromatography 83.1 Alkaloids 83.2 Amines 103.3 Antibiotics 133.4 Barbiturates 143.5 Carbonyl Compounds and Car-

boxylic Acids 143.6 Catecholamines 163.7 Hydroxy Compounds 163.8 Steroids 173.9 Sulfur Compounds 18

4 Capillary Electrophoresis 194.1 Derivatization for Ultraviolet

Detection 194.2 Derivatization for Fluorescence

Detection 204.3 Special Applications 214.4 Derivatization Modes 22

5 Conclusion 23

Abbreviations and Acronyms 23

Related Articles 23

References 24

This article describes both pre- and postcolumn deriva-tization chemistry used in conjunction with either chro-matography or capillary electrophoresis (CE) to facilitatethe determination of drugs. Generally, only prederivat-ization is used in gas chromatography (GC), principally

to enhance the volatility, temperature stability, and/ordetectability. The GC section considers derivatization ofdrugs by reagent class: alkylation, acylation, and silylation.The GC sample handling section describes an approachwhich combines the extraction and derivatization stepstogether. Both pre- and postcolumn derivatization arecommon approaches for high-performance liquid chro-matography (HPLC) and many of these methods havebeen adapted for CE. Derivatization for HPLC is oftendirected toward aliphatic amines, carboxylic acids, or alco-hols that are difficult to detect at low levels by absorbance,luminescence, or electrochemical means. In addition, smallhydrophilic molecules upon prederivatization are oftenconverted into larger more hydrophobic compounds,making reversed-phase HPLC easier or even feasible.The HPLC section considers derivatization of drugs basedon type: alkaloids, amines, antibiotics, barbiturates, car-bonyl compounds and carboxylic acids, catecholamines,hydroxy compounds, steroids, and sulfur compounds. Forthe GC and HPLC sections, tables are included givingthe structures of the more important derivatizing agents,the analytes, and the corresponding reaction products. Abrief rationale for derivatization chemistry with CE con-cludes this article. For CE, derivatization is often done toimprove detectability since the path length for absorbancedetection is very short and fluorescence can be effectiveusing laser-based systems.

1 INTRODUCTION

Derivatization in conjunction with chromatography orCE can be done either in an off-line mode, often priorto the separation step (prederivatization), or in an on-line mode (postcolumn derivatization). Generally, onlyprederivatization is used in GC, principally to enhancethe volatility, temperature stability, and/or detectability.Both pre- and postcolumn derivatization are commonapproaches for HPLC and many of these methodshave been adapted for CE. Derivatization for HPLCis often directed toward aliphatic amines, carboxylicacids, or alcohols that are difficult to detect at lowlevels by absorbance, luminescence, or electrochemicalmeans. In addition, small hydrophilic molecules uponprederivatization are often converted into larger morehydrophobic compounds, making reversed-phase HPLCeasier or feasible. For CE, derivatization is often done toimprove detectability since the path length for absorbancedetection is very short and fluorescence can be effectiveusing-laser based systems.

The desirable conditions to be met for precolumnderivatization are (1) the reaction stoichiometry andproduct structure should be known, (2) the reaction

Encyclopedia of Analytical ChemistryR.A. Meyers (Ed.) Copyright John Wiley & Sons Ltd


2 PHARMACEUTICALS AND DRUGS

should be reasonably fast and proceed quantitatively (orat least reproducibly), and (3) the derivative should bestable and readily separable and distinguishable from thestarting material. One advantage of the prederivatizationapproach is that simple equipment is commerciallyavailable to allow for the reaction chemistry to be donein the batch mode. Subsequent sample analysis using anautoinjector and a standard unmodified chromatographis straightforward. Alternatively, the use of a robot tocarry out the entire prederivatization chemistry and anHPLC instrument equipped with an autosampler meansthat the sample throughput will usually be limited by thechromatographic step.

Because postcolumn derivatization involves mixing thecolumn effluent with a reagent or passing it througha reactor to form the derivative before it is detected,the automation step is built in. Sample handling beforeanalysis is minimal, reducing the chance of error fromsample loss. The conditions desirable to be met forpostcolumn derivatization are (1) although the reactionmust be rapid, within about 2 min, and be reproducible,the reaction products do not have to be very stable,(2) the reagent itself must have no or a very low detectionresponse, and (3) the reactor volume must be small tominimize dilution and band broadening.

The remainder of the article is divided into three majorsections, GC, HPLC, and CE. The GC section consid-ers derivatization of drugs by reagent class: alkylation,acylation, and silylation. In addition, a short discus-sion of sample handling is included. The HPLC sectionconsiders the derivatization of drugs based on type:alkaloids, amines, antibiotics, barbiturates, carbonyl com-pounds and carboxylic acids, catecholamines, hydroxycompounds, steroids, and sulfur compounds. A briefrationale for derivatization chemistry with CE concludesthis article.

2 GAS CHROMATOGRAPHY

Prior to analysis by GC, compounds containing functionalgroups with active hydrogens such as COOH, OH, NH,and SH need to be protected. Compounds with thesefunctional groups tend to form intermolecular hydrogenbonds, reducing volatility. They are also thermallyunstable and can interact with either fused silica orthe stationary phase, causing peak broadening. Severalgood review sources describing derivatization reagents,reactions, and applications for GC are available. At leastone commercial company provides a good overview of thecommercial reagents and the apparatus available..1/ Twoother references are fairly comprehensive in describingmany of the published methods for GC derivatizationeither by reaction class.2/ or type of organic compound..3/

Alkylation, acylation, and silylation reaction chemistriesare the most common approaches to derivatization forGC and will be the primary focus in this article. A specificexample and an overview for each of these reaction classeswill be described below.

2.1 Alkylation

Alkylation represents the replacement of an active hydro-gen, found in an organic acid or amine, by an aliphatic oraliphatic–aromatic (e.g. benzyl) group. Alkylation reac-tions can also be used to prepare ethers from alcoholssuch as phenol, thioethers from sulfur compounds, andN-alkylamines, amides, and sulfonamides from amines.One principal chromatographic use is the conversion ofcarboxylic acids into esters that are stable and volatilewith good chromatographic characteristics. Esterificationof carboxylic acids has traditionally been done throughreaction with an alcohol in the presence of an acid cata-lyst. Generally, a 1–2-mg sample of the acid is heated with100 µL of the alcohol (methanol or ethanol) containing3 M HCl for 30 min at 70 °C. The alcohol is evaporated,leaving the ester as a residue. A faster reaction in onlyabout 2 min using a boiling water-bath is possible witha BF3 –methanol solution (Table 1, reagent 1). Gener-ally the esters are extracted into n-heptane followedby evaporation of the solvent..4/ DMFDA is a rapidmethylating agent which provides quantitative yieldsupon mixing in a nonaqueous solvent with the sampleof interest (Table 1, reagent 2). Carboxylic acids are con-verted to methyl esters, thiols to thioethers, and phenolsto methyl ethers. Primary amines are converted intotheir N,N-dimethylaminoethylene derivatives and aminoacids are simultaneously modified as this derivative atthe amino group and as the methyl ester at the carboxylgroup. Aliphatic hydroxyl groups are not methylated.Ethyl, propyl, n-butyl, and t-butyl acetals may be usedanalogously.

Pyrolysis of a quaternary ammonium salt (QUAT) inthe presence of the organic analyte can be a convenientmethylation procedure..5/ Upon injection of the QUATmixed with the drug having a reactive amino, hydroxyl, orcarboxyl group, into the hot injection port (250–300 °C),formation of the appropriate methyl derivative willoccur (Table 1, reagent 4). The derivative is immediatelyswept onto the analytical column for separation anddetection. TMAH is the most common reagent used buttrimethyl (a,a,a-trifluoro-m-tolyl)ammonium hydroxidecan be used at a lower injection temperature, which isimportant for labile unsaturated fatty acids. This methodis particularly good for barbiturates, sedatives, xanthinebases, phenolic alkaloids, and dilantin.

PFB-Br can convert carboxylic acids, phenols, sulfon-amides, and thiols into halogenated derivatives that can



Table 1 Common GC derivatives – alkylation

No. Reagenta

FB: OF

F CH3

(BF3−methanol)

H1.

OCO

(CH3)2N

(DMFDA)

2. CH3

HCH3

3. FF

F CH2Br

FF(PFB-Br)

4.N+ −OH

(TMAH)

H3C

CH3

CH3

COOHR

COOHR

NH2R

OHR

SHR

COOHR

OHR

Sulfonamides

SHR

OHR

NH2R

COOHR

Compounds

COOCH3R

COOCH3R

NHR

OR

SR

CR

OR

SR

OR

NHR

COOCH3R

Derivatives

(CH2)2 N(CH3)2

CH3

CH3

O (PFB)O

(PFB)

SO2NHRHN(PFB)

(PFB)

CH3

CH3

a DMFDA, N,N-dimethylformamide dimethylacetal; PFB-Br, pentafluorobenzyl bro-mide; TMAH, trimethylanilinium hydroxide.

be easily detected by electron capture (EC) (Table 1,reagent 3). For carboxylic acids, the reaction is run in anorganic solvent in the presence of a base such as a tertiaryamine for 5 min at 40 °C. Interestingly, tertiary amineshave been derivatized with pentafluorobenzyl chlorofor-mate prior to GC with EC detection..6/ The analysis ofcannabidiol (CBD) and the internal standard tetrahydro-cannabidiol in blood plasma involved derivatization withPFB-Br followed by purification on a Florisil column..7/

The derivatization procedure involved refluxing a hex-ane extract with PFB-Br overnight with stirring. Acetonewashings of this solution were evaporated to dryness andthe residue was dissolved in hexane. A portion of thishexane solution was injected into a gas chromatographequipped with an EC detector. A typical chromatogramtaken on a packed OV-225 glass column is shown inFigure 1. Although a packed column has limited separa-tion capability, it can handle larger amounts of sample. Adetection limit of 50 ng mL�1 of plasma was possible.

2.2 Acylation

Acylation is the use of a carboxylic acid or carboxylic acidderivative to convert compounds that have active hydro-gens (OH, SH, NH) into esters, thioesters, and amides,respectively. Acyl derivatives tend to direct the mass

0

inj.

4 8

12

12

Time (min)16 24 28

Figure 1 Typical chromatogram from a plasma sample con-taining 2.0 µg of the internal standard tetrahydrocannabidiol (1)and calculated to contain 1.5 µg of the CBD (2). [Reproducedwith permission from Jones et al..7/]



spectrometry (MS) fragmentation patterns of compoundsproviding useful structural information. Perfluoroa-cylimidazoles such as trifluoroacetylimidazole (TFAI),pentafluoropropionylimidazole (PFAI), or heptafluo-robutylimidazole (HFBI) can react with hydroxyl groupsand primary or secondary amines to generate the halo-genated derivative and the relatively inert by-product imi-dazole (Table 2, reagent 1). Generally the reaction timesare 1–3 h at 75 °C. Indoleamines and indole alcohols,which are acid-sensitive compounds, have been modifiedby this procedure. Metoclopromide, an antispasmodicagent related to procainamide, has been derivatized withHFBI for GC with EC detection..8/ These reagents areoften used in bifunctional derivatization schemes involv-ing silylation chemistry. For example, phenolic aminescan be silylated with N-trimethylsilylimidazole (TMSI)to protect the hydroxyl group and acylated to protectthe amine group. Other halogenated imidazoles such asp-bromobenzyl- and p-chlorobenzylimidazole have beenused because the detectability of the EC detector isenhanced. However, the volatility and stability of thederivatives under GC conditions tend to be lower com-pared with the fluorinated types.

Table 2 Common GC derivatives – acylation

No.

1.

OCR′

2.

3.

OHR

OHR

Compounds

OR

OR

NN C

O

R′=CF3 (TFAI),

C2F5 (PFAI), or

C3H7 (HFBI)

C R′O

R1 NR1 C R′O

NHR2 R2

R RNH2 NH C R′O

O C R′O

R′=CF3 (TFAA),

C2F5 (PFAA), or

C3H7 (HFAA)

C R′O

OH O C R′O

NR1NHR2

R1 C R′R2 O

NHRNH2RCF3C N C CF3

OO

CH3

(MBTFA) NR1NHR2

R1 C CF3

R2 O

OROHR C CF3

O

C CF3

O

SRSHR C CF3

O

DerivativesReagenta

R′

a TFAA, trifluoroacetic anhydride; PFAA, pentafluoropropi-onic anhydride; HFAA, heptafluorobutyric anhydride; MBTFA,N-methylbistrifluoroacetamide.

MBTFA, shown in Table 2 as reagent 3, trifluoroacety-lates primary and secondary amines and carbohydratesunder mild nonacidic conditions..9/ For carbohydrates, asingle derivative for mono-, di-, tri-, and tetrasaccharideswas obtained. The N-methyltrifluoroacetamide by-product is stable and volatile, being amenable to GC.

Perfluoro acid anhydrides [R(CDO)�O�(CDO)R],such as TFAA, PFAA and HFAA, react readily withalcohols, phenols, and urine for the preparation ofperfluoroacyl derivatives, which are particularly usefulfor EC detection (Table 2, reagent 2). Triethylamine isadded as part of the reaction mixture to neutralize theacidic by-products and drive the reaction to completion.Drugs of abuse are often derivatized in this waybefore gas chromatography/mass spectrometry (GC/MS)confirmation. A GC/MS example of derivatization ofamphetamine and methamphetamine by HFAA.10/ isshown in Figure 2(a) and (b). The splitting of the peaks

050 000

5.50

6.00

6.50

7.00

7.50

8.00

8.50

9.00

9.50

10.0

0

10.5

0

12

100 000150 000200 000250 000300 000350 000400 000

(a) Time (min)

Abu

ndan

ce

1

2

050 000

5.50

6.00

6.50

7.00

7.50

8.00

8.50

9.00

9.50

10.0

0

10.5

0

100 000150 000200 000250 000300 000350 000400 000450 000500 000550 000

(b) Time (min)

Abu

ndan

ce

Figure 2 (a) Total ion current chromatogram of HFAA-deri-vatized amphetamine (1) and methamphetamine (2). Theoverloaded peak at about 5.6 min is due to excess HFAA.(b) Total ion current chromatogram of HFAA derivatizedamphetamine (1) and methamphetamine (2) after extractionof the excess derivatizing agent. The baseline level in thischromatogram is only about 10% of that in (a). The splittingof the drug peaks in both chromatograms is due to thepresence of the deuterated internal standards (amphetamine-d5and methamphetamine-d8). [Both figures reproduced withpermission from Jones and Mell..10/]



is due to the presence of deuterated internal standards.Excess HFAA, which causes a high background level,column contamination and eventual degradation of thestationary phase, was removed by the procedure outlinedbelow. The drugs of interest were extracted from 2 mLof urine by solid-phase extraction (SPE) columns with a10% 2-propanol in chloroform elution solvent. Sodiumperiodate oxidation of interfering drugs was carriedout if necessary. The extract was acidified with 10%HCl in methanol and the solvent evaporated beforereconstitution of the residue in hexane with 0.1 Mtrimethylamine. HFAA was added and the mixture washeated in a sealed tube for 1 h at 70 °C. After cooling,deionized water was added to the tube, the tube wasshaken, and then the aqueous phase was discarded. A4% ammonia solution was shaken with the remaininghexane volume and then this organic layer was removedfor injection into the GC/MS system. The limit ofquantification and limit of detection were both of theorder of 50 ng mL�1 and the linearity was observed up toat least 4000 ng mL�1.

2.3 Silylation

Silylation is probably the most widely used derivati-zation scheme for GC. Active hydrogens from acid,alcohol, thiol, amine, and amide groups can all beprotected, usually with trimethylsilyl (TMS) groups. Theease of silylation of these functional groups followsthe order alcohol > phenol > carboxylic acid > amine >amide. Steric hindrance is also a factor within a classof organic compounds. For alcohols, the reactivityorder is primary > secondary > tertiary, and, for amines,primary > secondary. In general, silylation reagentsare unstable and must be protected from moisture.Trimethylchlorosilane (TMCS) is the simplest reagentthat can be used, often to derivatize carboxylic acids(Table 3, reagent 1). TMCS is often used in conjunctionwith HMDS (Table 3, reagent 2) to improve the silylationof sugars and related compounds. GC of silyl derivativesis generally straightforward, with only one major concern,the presence of excess silylating agent. Often an excessof the silylating agent is used in the derivatization reac-tion to minimize the problem of moisture or other acidiccomponents in the sample. If possible, it is recommendedthat the excess silylation reagent be evaporated fromthe sample using a stream of nitrogen before injectioninto the GC column. This avoids several problems suchas large reagent blank peaks in the chromatogram andfouling of the flame ionization detector by SiO2 deposits.A polar stationary phase such as poly(ethylene glycol)(Carbowax 20 M) and free fatty acid phase (FFAP) willbe derivatized by excess silylation reagent and cannot beused.

Silylacetamides are represented in the most popu-lar group of silylation agents, owing to their abilityto react quickly and quantitatively under mild condi-tions. BSA is a highly reactive TMS donor (Table 3,reagent 3). BSTFA (Table 3, reagent 4) has the advan-tage of giving a more fluorinated by-product than BSA.This permits derivatization of lower molecular weightanalytes without potential overlap of the trifluoroac-etamide by-product. The addition of TMCS to BSTFAwill promote the derivatization of amides, secondaryamines, and hindered hydroxyl groups. A comprehen-sive article on drug detection methodology describesthe determination of cocaine, heroin, and metabolitesin hair, plasma, saliva, and urine samples after isola-tion by SPE and subsequent derivatization with BSTFAwith TMCS before analysis by GC/MS..11/ After spik-ing with internal standards, plasma, saliva, and hairextracts were passed through SPE columns and the drugseluted with a methylene chloride–2 propanol–ammoniasolvent. The eluate was evaporated to dryness, recon-stituted with acetonitrile, and allowed to react with aBSTFA–TMCS mixture at 60 °C for 30 min before analy-sis by GC/MS. Representative chromatograms are shownin Figure 3(a–c). Separations were effected on a rel-atively short 12 mð 0.2 mm ID capillary column. Forurine, saliva, and plasma, the limit of detection was about1 ng mL�1 and for hair 0.1 ng mg�1.

MSTFA has similar donor strength to BSA andBSTFA but generates an even more volatile by-product,N-methyltrifluoroacetamide (Table 3, reagent 5). Bothexcess MSTFA and the by-product often elute with thesolvent peak, eliminating the presence of extra peaksin the chromatogram. Cocaine and its metabolites benz-oylecgonine and ecgonine methyl ester were sequentiallyderivatized with thyliodide to obtain the ester derivativeand then MSTFA to form the o-TMS derivatives..12/

The derivatized compounds were separated by a GCinstrument with a methylphenylsilicone column andnitrogen–phosphorus detection. Again TMCS can beadded to MSTFA to promote the derivatization of amidesand hindered amines and hydroxyl groups.

The strongest reagent for silylation of hydroxyl groupsis TMSI (Table 3, reagent 6). It does not react with aminesor amides and is effective for most steroids, includingthose with unhindered and highly hindered OH groups.It also reacts quickly and smoothly with carboxyl groups.

Silyl compounds other than those that derivatizewith a TMS group have also been used in con-junction with GC. Methyltestosterone was deriva-tized using either the dimethylethylsilyl (DMES)-imidazole or dimethylisopropylsilyl (DMIPS)-imidazoleto the corresponding silyl ether derivative..13/ Reac-tion conditions involve heating at 70 °C for 1 h beforeremoval of the excess reagent by N2 and reconstitution



Table 3 Common GC derivatives – silylation

No.

1. COOHR

Compounds

H3C SiCH3

Reagenta

CH3

Cl(TMCS)

2. OHRH3C Si

CH3

NCH3

(HMDS)

Si CH3

CH3

CH3

OR Si(CH3)3

CR O Si(CH3)3

O

3. OHR

H3C SiCH3

OCH3

(BSA)

C

OR Si(CH3)3

NR Si(CH3)3

Si(CH3)3

NH2R

NR1 Si(CH3)3

R2

NHR1

R2

OC

OC NH2R R N Si(CH3)3

Si(CH3)3

OCCOOHR R O Si(CH3)3

4.H3C Si

CH3

O

CH3

(BSTFA)

C N

CF3

Si CH3

CH3

CH3

Same as BSA, leaving group

F3C C NO

Si (CH3)3

H

more volatile than that of BSA

5.F3C

(MSTFA)

C N Si CH3

CH3

CH3

Same as BSA, leaving group

F3C C NO

CH3H

is very volatile

H3C

O

6.N Si CH3

CH3

CH3

N

(TMSI)

ROHR O Si(CH3)3

(steroids)

RCOOHR C O Si(CH3)3

O

No reaction with amines

H

N Si CH3

CH3

CH3CH3

Derivatives

a HMDS, hexamethyldisilazane; BSA, N,O-bis(trimethylsilyl)acetamide; BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; MSTFA, N-methyl-N-trimethylsilyltrifluoroacetamide.

of the residue in cyclohexane. Because the desiredapplication was the assay of methyltestosterone inbulk powder and tablets, separation on a packedcolumn was adequate in the concentration range0.1–1.5 mg mL�1 (Figure 4a–c). Alternatively, N-methyl-N-(t-butyldimethylsilyl)trifluoroacetamide (MTBSTFA)will derivatize hydroxyl, carboxyl, thiol, primary andsecondary amines by adding t-butyldimethylsilyl(TBDMS) groups. These TBDMS derivatives are morestable to hydrolysis than TMS compounds and, when ana-lyzed by GC/MS, a strong M� 57 fragment is noted which

can be used to determine the original compound’s molec-ular weight. These advantages have been demonstratedin the determination of short- and long-chain carboxylicacids..14/ Pentafluorophenyldimethylsilyl (flophemesyl)derivatives are often generated from steroids by reactionwith flophemesylamine at room temperature for 15 min.This is a selective reaction with only primary andsecondary hydroxyl groups in steroids reacting in the pres-ence of unprotected ketone groups. GC with EC detectionprovides detection limits in the nanograms–picogramsrange. Flophemesyl derivatives also have favorable



advantages for GC/MS, producing diagnostic ions whichcarry more of the current than TMS derivatives..15/

2.4 Sample Handling

Generally, drugs are found in aqueous matrices such asurine and plasma; extraction with an organic solvent is

12

3

4

5

6

7

89

9

10

11

12

13

14

14

15

16

16

17

18

19

19

20

20

21

2223

23

24

2

2

34

6

7

8

9

5

1

5

7

(a)

(b)

4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0

(c) Time (min)

10

11

12

15

14

17

19

20

21

23

2416

×75 ×5 ×1 ×5 ×75 ×15

Figure 3 Single ion monitoring recordings of extracts from(a) standard cocaine/opiate hair, (b) drug-free control hair,and (c) a hair sample collected from a heroin user. Ana-lytes are identified as follows: anhydroecgonine methyl ester(1), [2H-3]ecgonine methyl ester (2), ecgonine methyl ester(3), ecgonine ethyl ester (4), [2H-3]cocaine (5), cocaine (6),[2H-3]cocaethylene (7), cocaethylene (8), [1H-3]benzoylecgo-nine (9), benzoylecgonine (10), norcocaine (11), norcoceathy-lene (12), benzoylnorecgonine (13), [2H-3]codeine (14), codeine(15), [2H-3]morphine (16), morphine (17), norcodeine (18),[2H-6]-6-acetylmorphine (19), [2H-3]-6-acetylmorphine (20),6-acetylmorphine (21), normorphine (22), [2H-9]heroin (23),heroin (24). [Reproduced with permission from Wang et al..11/]

2 4

Time (min)

6

1

2

2980

2880

2960

3060

(c)

2

1

(a)

(b)

27702840

1 2R1

CH2H

R2

CH3C2H5

(1)(2)

R1

N

R2

OCH 3

Figure 4 Typical gas chromatograms of (1) methyltestosteroneand (2) norethandrolone as (a) TMS, DMCS (b) and (c)DMIPS derivatives. Note the bulkier derivatives are shiftedto a cleaner area in the GC trace. [Reproduced with permissionfrom Zakhari et al..13/]

required not only to isolate the drug in a nonaqueoussolvent which is compatible with most derivatizationreagents but also to remove inorganic salts whichare not compatible with GC. An alternative approachcalled direct derivatization combines the extraction andderivatization steps together. Direct derivatization ofdrugs in untreated biological samples for GC analysishas the primary advantages of improved extractability of



a derivatized polar compound and avoidance of stabilityproblems. Direct derivatization of drugs in untreatedbiological samples can mean not only derivatization inthe sample matrix followed by extraction, but also a two-phase reaction where the derivatization takes place in theorganic phase while extraction of the analyte is continuingfrom the aqueous phase..16/ Extractive alkylation hasbeen applied to valproic acid and ketoprofen, in whichthe tetrabutylammonium ion acted as the anion-pairingagent to pull the compound into the organic phase, wherealkylation to generate the methyl or phenacyl derivativewas possible. Phenolic compounds such as elioquinolhave been determined in an analogous fashion. Acylationinvolving perfluorinated anhydrides and chloroformateshas also been used for the determination of drugs such asmetanephrine and normetanephrine. Chloroformates cande-alkylate tertiary amines to form stable carbamates.

3 HIGH-PERFORMANCE LIQUIDCHROMATOGRAPHY

The variety of chemistries that can be adopted foreither pre- or postcolumn derivatization in conjunctionwith HPLC is large in part because either aqueousor organic solvent-compatible reactions are possible.Most of the books reviewing fundamental reactionsfor pre- and postcolumn derivatization chemistry inconjunction with HPLC were published in the 1980s,which appears to have been the most active time forresearch in this field..17 – 23/ Many review articles havefocused on postcolumn derivatization with HPLC..24 – 31/

Some specific review articles on derivatization chemistryof pharmaceutical compounds with HPLC have also beenpublished..32 – 36/ Although the other articles do not try tobe comprehensive, Ahuja.35/ gave a virtually completereview of the topic up to 1979. Danielson et al..36/

published another fairly complete review covering theperiod 1979–87, which is the basis for this part of thisarticle. Again, a specific example and an overview for eachmajor class of pharmaceutical compounds will be given.

3.1 Alkaloids

Separation of atropine and ergotamine by normal-phaseliquid chromatography (LC) followed by postcolumnion-pair extraction with an aqueous solution of 9,10-dimethoxyanthracene-2-sulfonate has been reported..37/

The organic mobile phase was monitored fluori-metrically with detection limits of 40–100 ng. Con-versely, methadone, phencyclidine, and their metabo-lites were separated by reversed-phase HPLC using9,10-dimethoxyanthracene-2-sulfonic acid in the mobilephase..38/ The resultant ion pairs (Table 4, reagent 3)

were extracted with chloroform postcolumn on-lineand detected fluorimetrically with detection limits of1–6 ng mL�1 in plasma. A precolumn ion-pair derivatiza-tion method for atropine, hyoscyamine, scopolamine, andergotamine, involving picric acid and normal-phase chro-matography, provided ultraviolet (UV) detection limitsof about 200 ng..39,40/

Morphine has been detected fluorimetrically afterpostcolumn oxidation with alkaline potassium hexa-cyanoferrate(II) to form the dimer pseudomorphine..41/

Morphine can be oxidized to the fluorescent dimer pseu-domorphine using alkaline hexacyanoferrate(III). Otheropiates, such as normorphine, naborphine, codeine, nor-codeine, and others, were also reactive. The excitation andemission wavelengths were 323 and 432 nm, respectively.A comparison of the UV and fluorescence response wasmade using the instrumentation shown in Figure 5. Themobile phase, a 12.5 : 87.5 methanol–0.1 M KBr solution,was propelled through a C18 reversed-phase column. Thederivatizing agent was 50 mg of K3Fe(CN)6 in 250 mLof 4 M ammonia solution delivered at 0.4 mL min�1. Thepresence of 4% of the nonionic surfactant Triton X-100 at the micellar level in the derivatization reagentsolution increased the signal about twofold. The reasonfor the improvement was credited to an increase in theactual dimer formation yield. Morphine could be deter-mined at the 2–30 µg mL�1 level in biological samples.The selectivity advantage of the fluorescence detector forthe determination of morphine in a urine extract wasshown and urine and serum samples were analyzed withdetection limits of about 10 ng. Fluorescence detection ofmorphine and related opiates was improved after postcol-umn derivatization with alkaline hexacyanoferrate(III)and micelle formation with Triton X..42/ A detection limitof 0.2 pmol was possible for morphine after precolumndansylation..43/ The heroin metabolite 6-acetylmorphinehas been determined in urine by reversed-phase HPLCwith fluorescence detection after automated precolumnoxidation with hexacyanoferrate(III)..44/ A detectionlimit of 1 ppb was reported. Reserpine, an antihyperten-sive agent, was detected fluorimetrically after postcolumnreaction with nitrous acid and UV irradiation..45,46/ Usingthis UV photochemical reaction, a 20-fold increase insignal over the native fluorescence of reserpine was found.

Physostigmine, an acetylcholinesterase inhibitor, wasseparated from its degradation products and reacted withcoulometrically generated bromine..47/ Electrochemicaldetection of unreacted bromine was inversely propor-tional to the amount of drug, and a detection limitof 0.5 ng could be attained. An on-line photochemi-cal reaction detector caused decreased fluorescence ofergot alkaloids, permitting the identification of these com-pounds in complex chromatograms..48/ A similar systemhas been shown to convert cannabinol into a fluorescent



Table 4 Common fluorescent (F) and electrochemical (E) derivatives for HPLC

No.

1.

Reagent(s) DerivativeCompounds

R′

CHOCHO

, R′′SH N

S

R′

R′′

2. RNH2 (F) N

CN

RCHO

CHO, CN−

3. R3NH+ (F)SO3

−OCH3

OCH3

SO3− +NHR3

OCH3

OCH3

4. RSH (F)NN

CH3H3C

BrH2C CH3

OO

NN

CH3H3C

RSH2C

OO

5. RCOOH (E) OHH2N OHRCHNO

6. Steroid (E) NO2H2NHN NO2NHN

OHHO

O

NH2 (F)

Derivatizationreagentreservoir

Waste

PumpII

Fluorescencedetector

Gauge

Waste

3-Wayvalve

Tee

Reactioncoil

Dual-penrecorder

UVdetector

ColumnInjector

PumpI

Mobilephasereservoir

Figure 5 HPLC system for morphine using postcolumn derivatization and fluorescence detection. [Reproduced with permissionfrom Nelson et al..41/]



derivative providing detection limits of less than 1 ngin urine..49/ Post-column photochemical derivatizationhas the advantage of no sample handling and withoutthe problems of pumping a reagent or detection back-ground from a reagent. On-line reaction of cannabinoidswith Fast Blue Salt B produced colored derivatives fordetection at 490 nm..50/ Pilocarpine was quaternized withp-nitrobenzyl bromide before reversed-phase HPLC sep-aration and detection at 254 nm..51/

3.2 Amines

Primarily fluorescent methods have been developed foramino acids and peptides. The o-phthalaldehyde (OPA),mercaptoethanol (or other thiol) reaction (Table 4,reagent 1) has been well studied in both pre- andpostcolumn modes. Specific articles describing com-mon amino acids are cited in general references..17 – 20/

Other amino acids such as s-carboxymethyl-L-cysteine,.52/

baclofen,.53,54/ and melphalan.55/ have also been deriva-tized with OPA. A more recent modification of thismethod is to use naphthalene-2,3-dicarboxyaldehyde withcyanide ion (Table 4, reagent 2)..56/ Detection limits downto 200 fmol with improved stability of the derivatives werestated advantages. g-Aminobutyric acid was modifiedwith dansyl chloride before reversed-phase HPLC and flu-orescent detection..57/ Detection at 360 nm was possiblefor N-acetylcysteine after reaction with 2,4-dinitro-1-fluorobenzene..58/ 1,2-Diamino-4,5-dimethoxybenzenewas used to form a fluorescent derivative of p-hydroxybestatin..59/ The peptide leupeptin was reactedthrough the guanidino moiety with benzoin to form a flu-orescent derivative..60/ Felypressin, a nonapeptide, wasderivatized with fluorescamine, providing detection li-mits of 0.3 ng..61/ A dinitrophthalic anhydride reaction ofpeptides permitted either electrochemical or absorbancedetection..62/ An ion-pair detection technique has beenapplied to hydrophobic amino acids and peptides..63/

A variety of other fluorescent methods have alsobeen reported for specific drugs. Tranexamic acid hasbeen detected using OPA..64/ Biogenic amines suchas tyramine, tryptamine, and serotonin were reactedwith OPA in a post-.65/ or precolumn.66/ mode. TheOPA precolumn fluorescent reaction has also beenapplied to L-buthionine-(S,R)-sulfoximine in plasma.67/

and mexiletine..68/ Fluorescent derivatization with flu-orescamine has been applied to tocainide.69/ and inthe postcolumn mode for sulfapyridine..70/ Enantiomersof mexiletine have also been resolved..71/ After con-version of the drug panthenol to aminopropanol, thefluorescamine reaction provided detection limits of0.4 µg..72/ The determination of debrisoquine and itshydroxy metabolites was possible by reaction throughthe guanidine moiety to form fluorescent compounds..73/

A postcolumn photochemical reactor caused the cleav-age of methotrexate to form the highly fluorescent2,4-diaminopteridine-6-carboxyaldehyde..74/ Indolethyl-amines were condensed with an aldehyde or a-keto acidto form fluorescent carboline derivatives..75/ Thiamine(vitamin B1) was oxidized with hexacyanoferrate(III)in base to give the fluorescent thiochrome in eitherthe precolumn.76/ or postcolumn.77/ mode. Riboflavin,already fluorescent, and thiamine, after precolumn oxi-dation to thiochrome using hexacyanoferrate(III), weredetermined by HPLC in a variety of food powders suchas flour, milk, and beans..78/ Hydrazine compounds, suchas isoniazid, were reacted with m-fluorobenzyl chlo-ride before reversed-phase separation and detection at220 nm..79/ A precolumn coumarin reaction permitted flu-orescent detection of 5-fluoro-20-deoxyuridine..80/ Fluo-rescein isothiocyanate (FITC) modified a bronchodilatorbefore HPLC–fluorescence analysis..81/

Some UV methods have been developed for aliphaticamines. Phenyl isothiocyanate has been used as a precol-umn derivatizing agent for amino acids in conjunctionwith reversed-phase HPLC. Both primary and sec-ondary amines can react (Table 5, reagent 1B) and thearomatic tag provides good retention for the smalleramino acids..82/ Both primary and secondary amineswere derivatized with 9-fluorenylmethyl chloroformate(FMOC) before separation by reversed-phase HPLCwith UV detection at 254 nm. After SPE of a urine sam-ple, b-phenethylamine was determined with a detectionlimit of 0.1 ng mL�1..83/ Amphetamine and metham-phetamine have been reacted with 4-methoxybenzoylchloride (Table 5, reagent 1A) and other acid chlo-rides for subsequent UV detection..84/ A comparisonof methods for amphetamines has been made..85/ Thecardiotonic drug heptaminol has been derivatized withaminoazobenzene-4-isothiocyanate for UV detection at420 nm.86/ or OPA for electrochemical detection..87/ Sec-ondary amines, such as piperazines, were converted intoUV derivatives with m-toluoylacyl chloride..88/ Enan-tiomers of metoprolol have been separated after reactionwith the chiral reagent (S)-(�)-phenylethyl isocyanate..89/

The determination of isophenindamine in the presenceof phenindamine tartrate has been achieved by formingcharge transfer complexes using AgNO3 and reversed-phase HPLC..90/

Antihistamines and other pharmaceuticals with atertiary amine moiety have been derivatized to pro-vide for luminescence detection. An ion-pair extrac-tion detector using dimethoxyanthracene sulfonate(Table 4, reagent 3) has permitted the fluorescent deter-mination of tertiary amines such as bromopheni-ramine and chlorpheniramine,.91,92/ ephedrine,.93/ andhyoscyamine..94,95/ Detection limits of 200–500 pg werepossible. Precolumn reaction of chlorpheniramine with



Table 5 Common UV derivatives for HPLC

No.

1.

Reagent(s) DerivativeCompounds

RNH2C

Cl

OCH3

O(A)

(B)NCS

CN

OCH3

O(A)

(B)NHCSNHR

R

H

2. R2NH SO2Cl SO2NR2

3. R3N

OC

Cl

O(A)

(B)C C N CH2 H2

CH

NH

CNHR2

O

4. RCOOH

CH2BrCO

Br

CCO

Br

OC

R

OH2

5. RSH ClCl

OCH2COOHCO

CC2H5

H2C

ClCl

OCH2COOHCO

CHC2H5

H2CRS

6. Penicillin

N

SN

O

CH3

CH3

COOH

CR

O

HNaOH, HgCl2, EDTA

−OOC N

SN

CH3

CH3

COO−

CR

O

H

H

CH2

O

benzyl chloroformate gives a fluorescent derivative witha detection limit of 0.1 ng mL�1..96/ Antihistamines, suchas diphenhydramine, were converted through the tertiaryamine group into fluorescent derivatives using 2-naphthylchloroformate..97/ Aliphatic tertiary amines can be deter-mined at the picomole level by postcolumn chemilumi-nescent detection, using tris(bipyridyl)ruthenium(III)..98/

Antihistamines in urine have also been separated byHPLC and detected with good selectivity using this ruthe-nium metal complex (Figure 6a–c)..99/ Tamoxifen and itsmetabolites in human serum can be UV photochemicallyactivated to form fluorescent phenanthrenes..100/ Post-column UV irradiation of tamoxifen and its derivatives

caused rearrangement to a substituted phenanthrene,permitting fluorescent detection with 0.1 ng mL�1 detec-tion limits..101/

Derivatives of nicotine, cotinine, and other metabo-lites can be detected at 530 nm in urine after HPLC,.102/

by using diethylthiobarbituric acid as a color-formingagent. Racemic phenothiazines were N-demethylatedwith vinyl chloroformate to form their secondary amines,which could then be reacted with (R)-(C)-1-phenylethylisocyanate (Table 5, reagent 3), and these compoundswere separated by reversed-phase HPLC..103/ Tertiarytetrahydroisoquinolines, such as diclofensine, were oxi-dized before photochemical conversion to fluorescent



AB

C

0 6 12 18 24

Time (min)(c)

A

BC

A

B

(a) (b)0 6 12 18 24 0 6 12 18 24

Figure 6 Chromatograms of an undiluted urine sample spikedwith (A) 0.15 µg mL�1 pheniramine, (B) 0.26 µg mL�1 bro-mopheniramine, and (C) 0.29 µg mL�1 diphenhydramine takenwith (a) UV (214 nm), (b) UV (254 nm), and (c) Ru(bpy)3

3C

chemiluminescence detection. [Reproduced with permissionfrom Holeman et al..99/]

isoquinolinium derivatives..104/ After postcolumn ion-pairextraction of secoverine, peroxylate chemiluminescencedetection was carried out..105/

Postcolumn detection of platinum(II) antineoplasticagents such as cisplatin is possible, as shown in Figure 7.The cisplatin-derived species are first reacted with aoxidant such as dichromate to form an activated species,which then combines rapidly with bisulfate to give a UV-absorbing complex. Using knitted open-tubular reactors,delay times of 26 s for the dichromate reaction and 4.7 minfor the bisulfate reaction were found to be optimal.Figure 8(a) and (b) shows the improvement in responseover conventional UV detection using the derivatization

Pump 1

Detector

λ = 290 nm

Pump 3

∆ t1

∆ t2

Mobile phase(0.7 mL min–1)

Pump 2

K2Cr2O7

(0.1 mL min–1)

NaHSO3

(0.1 mL min–1)

Analyticalcolumn

Figure 7 Schematic representation of the Pt�HSO3� reaction

detector for cisplatin-type compounds. [Reproduced withpermission from Marsh et al..106/]

(a) Time (min)

0 5

12

3

Abs

orba

nce

(300

nm

)

0.00

25 a

u

(b) Time (min)

0 5 10

1

23

Abs

orba

nce

(290

nm

)

0.05

au

Figure 8 Comparison of UV detection (300 nm) after HPLCseparation (a) and reactor response (290 nm) to derivatizedplatinum (b) for a mixture of cis-dichloroplatinum com-plexes: (1) cis-dichloro(1,2-diaminocyclohexane)platinum(II),(2) cis-dichloro(ethylenediamine)platinum(II), and (3) cis-di-chlorodiammineplatinum(II). [Reproduced with permissionfrom Marsh et al..106/]

scheme. Detection limits of 5–10 µg mL�1 were possible.Cisplatin was also determined in a plasma ultrafiltratesample at the 5-ng level..106/



3.3 Antibiotics

This class of pharmaceuticals has received major attentionfor both pre- and postcolumn derivatization. Fluores-cent derivatization of the primary amine group ofmany antibiotics with OPA and a sulfhydryl compound,often mercaptoethanol, has been commonly performed(Table 4, reagent 1). Gentamicin,.107,108/ penicillin V afterenzymatic conversion to 6-aminopenicillanic acid,.108/

penicillin N,.109/ cephalosporin C,.110/ cycloserine,.111/

and fludalanine.112/ were assayed by using an OPApostcolumn reactor. Spectinomycin after postcolumnoxidation with hypochlorite could also be derivatizedwith OPA in a second reaction coil..113/ Reversed-phase separation was employed for all these separationsbecause the OPA reaction is carried out in an alka-line buffer solution. Precolumn reaction with OPA forgentamicin,.114/ sisomicin,.115/ phosphinothricin and itsalanine analog.116,117/ before reversed-phase separation,have all been reported. Amikacin isomers were firstadsorbed on a silica gel column before reaction withOPA; the derivatives were eluted with ethanol beforeseparation by reversed-phase HPLC..118/ A comparisonof pre- and postcolumn OPA reaction conditions forgentamycin has been made..119/ For all these OPA fluo-rescence methods, detection limits are about 1 µg mL�1.For example, b-lactams found in microbial fermenta-tion broths were separated on a C18 HPLC column anddetected fluorimetrically with excitation at 350 nm andemission at 450 nm after reaction with OPA and mer-captoethanol. Cephamycin, penicillin N, cephalosporinC, and 6-aminopenicillanic acid were separated in about12 min at a flow rate of 1.5 mL min�1 with postcolumnconditions involving the OPA reagent at pH 12, a reac-tion coil of 12 m, a temperature of 90 °C, and a reagentflow rate of 0.8 mL min�1. Detection limits of less than0.5 and 1.0 µg mL�1 were achieved for penicillin N andcephalosporin C, respectively..120/

Numerous other postcolumn UV, fluorimetric, andelectrochemical methods have been reported fortetracycline-type antibiotics and related compounds. For-mation of a mercuric mercaptide of penicillins permitsUV detection at 310 nm with detection limits of 10 ng..121/

Sulfonamides in egg, milk, and meat samples havebeen detected at 450 nm after derivatization with p-dimethylaminobenzaldehyde..122/ Monensin, narasin, andsalinomycin in animal feeds have been reacted withvanillin to give products detectable at 520 nm..123/ Peni-cillins, such as amoxicillin, ampicillin, and others, canbe separated by reversed-phase HPLC with alkalinedegradation in the presence of mercuric chloride.124/

(Table 5, reagent 6). Methanol promotes this reaction,forming the ring-cleaved product, which absorbs at274 nm, giving a detection limit of 50 ng mL�1. It was

discovered later that sodium hypochlorite could replacethe mercuric chloride reagent while maintaining a 1-minhydrolysis time..125/ Application of this latter method topenicillins in biological samples has been made..126,127/

Fluorescent detection of streptomycin reacted throughthe guanidino groups with naphthoquinone-4-sulfonate inalkaline solution has been reported in serum samples..128/

Photothermal derivatization of ciprofloxacin and itsmetabolites permits fluorescent detection throughout alinear range of about 2–1000 ng mL�1..129/ Photolysiswith electrochemical detection of penicillins and cefo-perazone has provided detection limits of about 6 ng..130/

Electrochemically generated bromine was used as an oxi-dizing agent for cephalosporins and their decompositionproducts..131/ The excess bromine was detected at 0.4 Vusing a glassy carbon electrode. A comparison of thismethod with UV detection and postcolumn fluorescaminederivatization has recently been summarized..132/ Acidi-fication of a serum sample sometimes improved recoveryof cephalosporins when micellar chromatography wasused with sodium dodecyl sulfate (SDS) in the mobilephase..133/ Fluorescamine has been used in an automatedsystem for amoxycillin in biological fluids..134/ Chemi-luminescence detection of clindamycin phosphate usingtris(bipyridyl)ruthenium(III).135/ gave detection limits of8 ppb compared with 970 ppb using UV detection at214 nm.

A variety of reagents are also available for precolumnderivatization of antibiotics and UV or fluorimetric detec-tion. Phenacyl esters of some natural penicillins wereprepared using dibromoacetophenone before reversed-phase HPLC with UV detection..136/ Nitrobenzenederivatives of neomycin B and C can be separatedby normal-phase HPLC and detected at 350 nm..137/

Similar methods using 1-fluoro-2,4-dinitrobenzene toform 2,4-dinitrophenyl derivatives of neomycin B andC.138,139/ and amikacin.140,141/ have also been published.Neomycin and other aminoglycosides have been pub-lished. Neomycin and other aminoglycosides have beenconverted to benzoyl derivatives before UV detection at230 nm..142/ Aminoglyosides have also been reacted with2,4,6-trinitrobenzenesulfonic acid, permitting UV detec-tion at 350 nm..143/ Ion-pair formation of methscopol-amine bromide with an aromatic anion during reversed-phase HPLC permitted UV detection..144/ The secondaryamine group of spectinomycin was derivatized with 2-naphthalenesulfonyl chloride (Table 5, reagent 2) beforenormal-phase HPLC and UV detection at 254 nm..145/

The reagent FMOC formed fluorescent derivatives ofnatural penicillins, cephalosporins, and their precursorsin biological samples..146/ b-Lactams were prederiva-tized with FMOC for 5 min at 20 °C in borate bufferat pH 7.7 before injection of the derivatives on toa C18 reversed-phase column. Excitation and emission



100

0

0 10 20 30 40 50

Retention time (min)

Rel

ativ

e flu

ores

cenc

e

Ace

toni

trile

(%

)

A

B

C

D

Figure 9 Reversed phase HPLC of FMOC derivatives ofcephalosporins (5 µg mL�1) in a fermentation broth usinga borate buffer–acetonitrile gradient mobile phase. (A)Deacetylcephalosporin C; (B) Deacetoxycephalosporin C;(C) cephalosporin C; (D) ampicillin. [Reproduced with per-mission from Shah and Adlard..146/]

wavelengths for the fluorescent detection were 260 and313 nm, respectively. A typical chromatogram is given inFigure 9 showing the modest acetonitrile gradient untilafter elution of ampicillin in which the acetonitrile is takento 100%. Detection limits were 0.01 and 0.05 µg mL�1

for 6-aminopenicillanic acid and isopenicillin N, respec-tively. Application of the method to fermentationbroths was made over a range of 0.05–100 µg mL�1..147/

A maleimide reagent gave a fluorescent derivativefor penicillamine..148/ An imidazole–mercuric chloridereagent can convert penicillins into mercury-stabilizedpenicillinic acids after reaction at 50 °C for 50 min prior toUV detection at 325 nm..149/ A comparison of this methodwith the postcolumn OPA fluorescence method has beenmade..110/ A similar method using 1,2,4-triazole andHg(II) for ampicillin, amoxicillin, and other antibioticshas been studied..150 – 152/ Two precolumn derivatizationstudies with UV detection were checked with real sam-ples. Tobramycin plus impurities neamine and kanamycinand also the degradation product nebramine were deriva-tized with 2,4-dinitrofluorobenzene for 20 min at 70 °C in0.8 mM sulfuric acid. Separation of the derivatives on aC18 column with detection at 365 nm was carried out andthe stability of tobramycin in ophthalmic solutions wasdetermined..153/

3.4 Barbiturates

Precolumn methods primarily include reaction withvarious alkylating reagents. The highly reactive chlorineof N-chloromethylphthalimides permits derivatization ofOH and NH functional groups to form UV-absorbingcompounds..154/ Detection limits of 5 ng for phenobarbitalhave been reported..154,155/

2-Naphthacyl bromide forms strongly absorbing deriva-tives of barbiturates at 254 nm with detection in plasmaor serum below the therapeutic range..156/ An on-line solid-phase anion-exchange extraction providedenhanced chromatographic selectivity of barbituratesin urine..157/ Postcolumn UV detection of barbituratescan be conveniently enhanced by mixing with a pH 10borate buffer..158,159/ A wavelength shift for maximumabsorbance from 220 to 240 nm provides for detectionlimits of about 6 µg for butabarbital.

The UV detection of barbiturates can be significantlyenhanced through postcolumn photochemical derivatiza-tion. Barbiturates have no significant absorbance above230 nm and detection in the 200–220-nm range can becomplicated by the presence of interfering peaks, par-ticularly in biological samples such as serum. Using a25 mð 0.25 mm ID Teflon knitted open tubular reactormounted around a mercury lamp, which provided an irra-diation time of about 190 s, excellent signal enhancementat 270 nm was possible for barbiturates (Figure 10a andb). It was determined by HPLC that de-alkylation at the5-position to give ethylbarbituric acid was the mechanismof the photochemical reaction..160/

3.5 Carbonyl Compounds and Carboxylic Acids

Most of these methods refer to carboxylic acids;short-chain carboxylic acids were modified using p-bromophenacyl bromide (Table 5, reagent 4) to formesters that could be easily detected by UV absorbance..161/

However, first a short summary of the methods involvingthe reduction of quinone compounds to form fluores-cent hydroxy derivatives will be given. Vitamin K1 inhuman plasma was electrochemically reduced at �0.4 Vand the fluorescent derivative detected at levels as low as25–50 pg..162/ Comparison of this method with UV andelectrochemical oxidation detection systems has beenmade..163/ Danthron (1,8-dihydroxyanthraquinone) hasbeen reduced with dithionite and determined by eitherflow injection analysis in Modane tablets or HPLC inurine..164/ A comparison of UV and fluorescence chro-matograms for danthron spiked in a urine sample showedthe selectivity advantage of fluorescence (Figure 11aand b). Dansylhydrazine formed fluorescent derivativesof tetraphenylacetone isomers before separation..165/

5-Bromomethylfluorescein was studied for the pre-derivatization of carboxylic acids for detection by either



0 10

Time (min)

Lam

p of

f

(a)

0 10

Time (min)

Lam

p on

(b)

3

1

2

5

4

Figure 10 Chromatogram of a standard sample of barbituratesdetected at 270 nm, (a) without and (b) with on-line photochem-ical reaction. (1) Aprobarbital; (2) butethal; (3) pentobarbital;(4) mephobarbital; (5) secobarbital. [Reproduced with permis-sion from Wolf and Schmid..106/]

UV absorbance or fluorescence (standard and laserinduced). Model analytes included prostaglandins (unsat-urated carboxylic acids) and the drug cefuroxime, and alsostandard aliphatic and aromatic carboxylic acids. Dicarb-oxylic acids did not react with bromomethylfluorescein,possibly owing to solubility problems in the organic reac-tion medium..166/ Fatty acids and prostaglandins wereconverted into p-hydroxanilides using p-aminophenol(Table 4, reagent 5). These hydroxy derivatives werethen oxidized through electrochemical detection afterreversed-phase HPLC..167/ A UV-absorbing naphthacylester of the prostaglandin carboprost has been formedbefore normal-phase HPLC..168/ Fluorescent derivativesof prostaglandins using 9-anthryldiazomethane pro-vided detection limits of 100 pg after reversed-phaseHPLC..169/ The prostaglandin arbaprotstil was deriva-tized with panacyl bromide before column switching usingfluorescent derivatization..170,171/ Prostaglandins havealso been derivatized with p-(9-anthroyloxyl)phenacylbromide..172/ Oxidation of prostaglandins to the corre-sponding 15-oxo derivatives using pyridinium dichromate

0

(a)

62 4

Time (min)8

Danthron

0.04 AUFS

UV

(b)

40

Time (min)8

0.15%Relative

fluorescence

Figure 11 Separation of danthron spiked in urine (2 ng mL�1).(a) UV detection (254 nm). Column, 250ð 4.6 mm C18; mobilephase, 60 : 40 methanol–water at 1.2 mL min�1. (b) Fluorescencedetection (388 nm excitation, 510 nm emission). A solution of90 mM dithionite in 2% borate buffer was pumped into theeffluent at 0.4 mL min�1. [Reproduced with permission fromMiller and Danielson..164/]

permitted UV detection at 228 nm and picomole detectionlimits..173,174/ Prostaglandins have been labeled with thefluorescent reagent 9-anthoyldiazomethane through thecarboxyl group. This esterification reaction can be carriedout under mild conditions such as 40 °C for 30 min. Fiveprostaglandin derivatives were separated by reversed-phase HPLC with fluorescent detection with excitation at365 nm and emission at 418 nm at the 8-ng level..175/

Ascorbic acid from 5 to 800 mg L�1 was detectedby chemiluminescence after postcolumn reaction withlucigenin..176/ After derivatization with 1,2-phenylenedia-mine both ascorbic acid and dehydroascorbic acid wereseparated by reversed-phase ion-pair HPLC..177/ Thesame two compounds were separated by reversed-phaseHPLC, and the dehydroascorbic acid was reduced toascorbic acid with dithiothreitol for UV detection at267 nm..178/ UV detection of bis(dinitrophenyl)hydrazinederivatives of ascorbic and dehydroascorbic acid was



possible at 497 nm..179/ Four forms of ascorbic acid,the previous two plus isoascorbic acid and its dehy-dro form, were detected postcolumn using benzami-dine and fluorescence..180/ Total ascorbic acid wasdetermined fluorimetrically after reaction with diamino-dimethoxybenzene..181/

Finally, a variety of methods directed toward painrelievers and other compounds have been published.Indomethacin formed a fluorescent derivative after post-column alkaline hydrolysis, giving a detection limit of5 pg..182/ Postcolumn alkaline hydrolysis has also beenapplied to aspirin in plasma in order to form the fluores-cent salicylic acid..183/ The enantiomeric composition ofibuprofen in human plasma has been resolved after pre-derivatizationwith(S)-(�)-1-(naphthenyl)ethylamine.184/

or ethyl chloroformate–leucinamide..185/ Flunoxaprofenenantiomers have been separated after reaction with (S)-(�)-1-phenylethylamine..186/ Artesunic acid was deter-mined after derivatization with o,p-nitrobenzyl-N,N0-di-isopropylisourea by HPLC with UV detection..187/ Cho-line, betaine, and related compounds have been reacted toform either 40-bromophenacyl esters.188/ or p-nitrobenzyloxines.189/ to permit UV detection at 254 nm. A newacridinium sulfonylamide label permits the chemilumi-nescent detection of carboxylic acids such as the testcompound ibuprofan. This alkylation reaction takes placein dry acetonitrile for 20 min at 50 °C and separation ofthe derivative is possible on a C18 column with an ace-tonitrile–water–tetrahydrofuran mobile phase with theion-pairing agent tetrabutylammonium ion. Chemilumi-nescence detection of the acridinium label is possibleby postcolumn addition of potassium hydroxide solution.A detection limit of 3 pg of derivatized ibuprofen wasfound..190/

3.6 Catecholamines

Precolumn fluorescent derivatization of catecholamineswith OPA and a thiol has been well established(Table 4, reagent 1). Norepinephrine, dopamine, andnormetanephrine have been measured at the lowpicogram level after reversed-phase HPLC..191,192/ Anelectrochemical cell placed between the injector andcolumn was used for oxidation of adrenaline and lev-odopa to the corresponding quinones for detectionin the visible region..193/ Resolution of norephedrineenantiomers after derivatization with either acetylgly-cosyl isothiocyanates.194/ or 4-methyl-5-phenyl-2-oxazoli-done.195/ has been reported. Fluorescent catecholaminederivatives have been generated using 1,2-diphenylethyl-enediamine, providing detection limits in the femtomolerange..196/

Postcolumn fluorescent reaction of norepinephrine andepinephrine with trihydroxyindole provided 1-pg detec-tion limits..197/ This method was compared with the

OPA–thiol reaction in the postcolumn mode..198,199/ Thesame two catecholamines can be converted into fluores-cent products by heating in an alkaline borate bufferafter ion-exchange chromatography..200/ Dopamine iso-mers in serum and urine have been separated beforehydrolysis and reaction with p-aminobenzoic acid toform fluorescent products..201/ Glycylglycine as an alter-native postcolumn reagent to glycinamide increasedthe rate of formation of fluorescent derivatives afterion-exchange HPLC..202,203/ Catecholamines catalyze thereaction between formaldehyde and o-dintrobenzene andpermit their detection at 560 nm..204/

3.7 Hydroxy Compounds

Most precolumn methods focus on enhancing UV detec-tion. Isotachysterol derivatives of vitamin D improveddetection at 290 nm after normal-phase HPLC..205/ On-column periodate oxidation of ephedrine sulfate, anasal decongestant, to form benzyl alcohol has beencarried out in a seven-laboratory study..206/ Indirectphotometric detection using n-heptyl p-aminobenzoatein the mobile phase and a C18 column has beenapplied to menthol..207/ Enantiomers of propranololand 4-hydroxypropranolol were formed upon derivat-ization with (C)-1-phenylethyl isocyanate.208/ or (C)-tetraacetyl b-D-glucopyranosyl isothiocyanate.209,210/ andseparated by reversed-phase HPLC. Enantiomers ofderivatized or underivatized propranolol have beenseparated..211/ Diastereomeric derivatization of 1-methyl-3-pyrolidinol.212/ and proxyphylline.213/ for UV detectionhas been accomplished. Qinghaosu, an antimalaria com-ponent in a Chinese herb, can be converted into aUV-absorbing compound using a sodium hydroxide solu-tion before HPLC separation..214/ A derivative of qing-haosu has been esterified with diacetyldihydrofluoresceinbefore HPLC and UV detection..215/ Cholic acid andits derivatives have been labeled with 1-anthroylnitrilebefore HPLC and fluorescent detection at the femto-mole level..216/ Trospium has been converted into thecorresponding spiro alcohol before fluorescent derivati-zation with benoxaprofen chloride and reversed-phaseHPLC..217/

Derivatization can be an effective method to facili-tate the chiral separation of pharmaceuticals. Nadololdiastereomers were derivatized with (R)-(�)-1-(1-naphthyl)ethyl isothiocyanate to chiral urea derivativesthrough the secondary amine group by reaction for 5 minat 45 °C. Separation of the RS, SR, RR, and SS diastere-omers was straightforward on a C18 column using a60 : 40 water–acetonitrile mobile phase (Figure 12a–c).Fluorescence detection with excitation at 285 nm andemission at 340 nm provided selectivity for the determi-nation of nadolol in plasma samples. The limit of detection



(a) 020406080

100120140160180200

(b) 020406080

100120140160180200

12 3 4

1

23

4

0

(c)

020406080

100120140160180200

5 10 15 20

Time (min)25 30 35 40 45

Figure 12 Typical chromatograms of (a) blank control dogplasma, (b) plasma spiked with 50 ng mL�1 of each dia-steromer and (c) plasma obtained 2 h after oral administrationof 1 mg kg�1 of racemic nadolol. Peaks: 1 D .SR/-nadolol;2 D .RS/-nadolol; 3 D .RR/-nadolol; 4 D .S,S/-nadolol. [Re-produced with permission from Hoshino et al..218/]

was 2.5 ng mL�1, representing 50 pg injected. This chiralderivatization method has been used for the determina-tion of enantiomers of other b-blocker drugs..218/

A few of the post-column approaches are outlinedbelow. Using a photochemical reactor, diethylstilbestrol(DES) has been converted into a fluorescent deriva-tive and determined at the low parts per billion levelin biological matrixes..219,220/ Anabolic stilbenes, such asDES, have been measured in urine by HPLC and an off-line chemiluminescence immunochemical assay..221/ Theantihypertensive agent fenoldopam was formed usinga postcolumn enzyme reactor from the correspond-ing glucuronide, and detected electrochemically..222/

Cyclodextrins in biological fluids were assayed bynegative colorimetric detection after postcolumn com-plexation with phenolphthalein..223/ Vitamin B6 (pyri-doxine) has been postcolumn derivatized with 2,6-dibromoquinone-4-chlorimide to form a colored productwith a maximum absorbance at 650 nm..224/

3.8 Steroids

Esterified estrogens were converted into their free phe-nolic forms by acid hydrolysis, and separation could beachieved by reversed-phase HPLC..225/ The chromato-graphic behavior of estrogen carbonyls, such as equilin,equilin, and estrone, was improved upon reduction to the17-a-hydroxy compounds using sodium borohydride..226/

Prederivatization of conjugated estrogens with dansylchloride permitted fluorescent detection after sepa-ration by normal-phase HPLC (Table 4)..227/ Keto-steroids, such as androsterone, dehydroepiandrosterone,epiandosterone, and etiocholanolone, were derivatizedwith p-nitrophenylhydrazine (Table 4, reaction 6) anddetected electrochemically at levels as low as 200 pg..228/

Isonicotinoyl hydrazine was used to tag steroids,such as corticosterone, to permit fluorescent detec-tion after normal-phase HPLC..229/ Further work usingthis method with reversed-phase HPLC and optimizedreaction conditions permitted detection limits as lowas 7–10 ng..230,231/ 17-Oxosteroids were labelled withdansylhydrazine and then chromatographed on a silicacolumn with subsequent fluorescent detection from 60 to100 pg..232/ A similar method for this class of compoundsusing 3-chloroformyl-7-methoxycoumarin and reversed-phase HPLC has been published..233/ Hydroxysteroidswere derivatized with anthroylnitrile, showing the fea-sibility of the reaction for fluorescent detection afterHPLC..234/

Six corticosteroids were separated within 25 minand reacted with a lucigenin–KOH solution forchemiluminescence detection..235/ The reactive site isan a-hydroxycarbonyl group, and detection limits of2 pg were comparable to those with precolumn fluo-rescent methods. Corticosteroids were also postcolumnderivatized with glycinamide in the presence of hexa-cyanoferrateIII) before fluorimetric detection at the 5-nglevel..236/ Digoxin, a widely used cardiac glycoside, hasbeen detected fluorimetrically by postcolumn reactionwith a solution of ascorbic acid, peroxide, and HCl..237,238/

A detection limit less than 1 ng was attained.Digoxin and its metabolites, such as digoxigenin, were

derivatized with 1-naphthoyl chloride before separationand fluorescent detection.239/ and 5-ng amounts could bedetermined in urine or feces. A similar method using4-nitrobenzoyl chloride has been reported..240/ Dihy-drodigoxin, a major metabolite of digoxin, has been



separated from digoxin with dual UV and postcolumnfluorescent derivatization detection..241/ The photoreduc-tion of anthroquinone-2,6-disulfonate to the fluorescent9,10-dihydroxyanthracene-2,6-disulfonate occurs only inthe presence of hydrogen atom-donating substrates, suchas alcohols, aldehydes, amines, ethers, and saccharides.Using a knitted Teflon reactor, cardiac glycosides, such asdigoxin, digoxigenin, and diginatin, have been determinedin the range 50–500 ng..242/

3.9 Sulfur Compounds

Most methods employ a precolumn reaction to formfluorescent or UV derivatives. Dansylaziridine.243/ wasused to determine cysteine and other thiols. Biologi-cal thiols, such as glutathione and ergothioneine, werereacted with monobromobimane (Table 4, reagent 4)before ion-exchange chromatography..244/ This methodwas also adopted for the determination of dithi-ols, such as 2,3-dimercaptopropane-1-sulfonic acid,in urine at levels down to 10 pmol..245/ Fluorescentderivatization of 2-mercaptopropionylglycine using N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide couldprobably be extended to other thiol compounds..246/

Resolution of the optical isomers of diltiazem was accom-plished using UV detection after derivatization withoptically pure 2-naphthylsulfonyl-2-pyrollidinecarbonylchloride..247/ Three N-substituted maleimides were com-pared for precolumn derivatization of thiols such aspenicillamine before electrochemical detection at thepicogram level..248/ Ethacrynic acid (Table 5, reagent 5)forms UV-detectable derivatives of thiols, such as capto-pril, N-acetyl-L-cysteine, and mercaptopropionylglycinewith detection limits down to 0.5 µg mL�1..249/

Postcolumn derivatization of thioethers, such as ampi-cillin and ranitidine, was accomplished using on-linegenerated bromine and electrochemical detection of theexcess bromine..250/ An analogous method has beenreported for phenothiazines, such as thioridazine; how-ever, the resultant products from the oxidation reactionwith bromine are detected fluorimetrically..251/ Photo-chemical activation of phenothiazines and demoxepamin 2 min and subsequent fluorescence detection pro-vided detection limits a factor of 10 better than UVdetection..252/ Applications of photochemical reactors toa variety of pharmaceuticals, such as phenothiazines, havebeen summarized..253,254/ A precolumn derivatizationmethod for phenothiazine involves desulfurization withRaney nickel to produce diphenylamine, which is elec-trochemically active..255/ A detection limit of 10 pg wasfound. The reagent pyrenemaleimide provided deriva-tization of N-acetylcysteine with a detection limit of10 pmol..256/ Postcolumn complexation of disulfiram andtwo of its metabolites using Cu2C allowed colorimetric

MP(B) P(B) C2

AS P(A) MP(A)

C1

CO

VAL1

MP(C)

DET(A)

DET(B)P(C)

C3

Waste

Waste

Waste

VAL2

Figure 13 Schematic diagram of the HPLC system. P(A),P(B), and P(C) D pumps; AS D autosampler; VAL-1 andVAL-2 D six-port valves 1 and 2; C0 D cleanup column; Cl,C2, and C3 D columns 1, 2, and 3; DET(A) and DET.B/ D UVdetectors A and B; MP(A), MP(B), and MP .C/ D mobilephases A, B, and C. The solid and dotted lines in thesix-port valves indicate valve positions 0 and 1, respectively.[Reproduced with permission from Funakoshi et al..259/]

detection at 435 nm with detection limits in the low partsper billion range..257/ Quenched peroxalate chemilumi-nescence has been employed using immobilized reagentsin a postcolumn reactor for thioridazin, sulforidazine, andmethimazole..258/

On-line precolumn derivatization to improve repro-ducibility has been demonstrated for the determinationof busulfan in human serum. A schematic diagram of theHPLC system is shown in Figure 13. All three columnsC1, C2, and C3 were C18 types with different lengthsand/or diameters. After extraction of busulfan fromserum, the residue reconstituted in water was injectedon to C1, where it was derivatized with diethyldithio-carbamate (DCC) in mobile phase A for 5 min. Thenthe busulfan–DCC derivative from C1 was backflushedon to C2, where it was separated using mobile phaseB. Because the background interference from mostlyexcess DCC overlapped completely the busulfan–DCCderivative peak, valve 2 was used to take a heart cutof this peak of interest and inject it on to C3. Theresulting separation using mobile phase C is shown inFigure 14(a) and (b). Detection was at 278 nm and thelower limit of quantitation in serum was 10 ng mL�1.The total time for the derivatization and separation was33 min..259/



0 8 16 24

Inj.

Time (min)(a)

Inj.

0 8 16 24

Time (min)0.

0006

a.u

.

(b)

Bus

ul fa

n/16

.150

Figure 14 Typical chromatograms of (a) drug-free serum and(b) serum spiked with busulfan (100 ng mL�1) obtained withcolumns Cl, C2, and C3. The arrow indicates the retention timeof the busulfan derivative. [Reproduced with permission fromFunakoshi et al..259/]

4 CAPILLARY ELECTROPHORESIS

CE has developed into a versatile separation techniquewell suited for the determination of pharmaceuticaland biomedical samples. The major advantages of CEover chromatographic separation techniques are itssimplicity and efficiency. However, CE suffers frompoor sensitivity in detection owing to the extremelysmall sample volume involved. One way to improve thesensitivity of CE detection is to derivatize the analyteswith more favorable detection characteristics by addingeither an ultraviolet/visible (UV/VIS) chromophore or afluorophore.

Derivatization in conjuction with GC and HPLCis a well-developed field. With the advent of CE,derivatization chemistry previously developed for HPLCis often applicable to CE..260/ At least one comprehensivereview specifically focused on the derivatization inCE has appeared..261/ Several other review articlesdealt with more specific topics, such as postcolumnderivatization,.262/ diastereomer derivatization,.263/ anddyes used to derivatize molecules for CE with laser-induced fluorescence (LIF) detection in drug analysis..264/

The pros and cons of the various modes of derivatizationand a detailed comparison of this topic between LC andCE can be found in the literature..261/ Instead of listingthe derivatization reagents suited for labeling amino,aldehyde, keto, carboxyl, hydroxyl, and sulfhydryl groups,we intend to focus on the purpose of derivatizationin CE from a practical point of view, i.e. enhancing

the detectability. Since UV/VIS and LIF detection arethe most common detection methods used in CE forpharmaceutical analysis, we shall discuss the most recentwork related to this area.

4.1 Derivatization for Ultraviolet Detection

Although most organic compounds have UV/VIS chro-mophores, derivatization is still important, and sometimesnecessary, for UV/VIS detection in CE. Very often,UV/VIS detection of the chromophores does not givea satisfactory response owing to the very short lightpathlength (50–100 µm) in CE. For example, the anti-cancer drug prospidin is a piperazinium derivative withchloroxypropyl groups but no UV/VIS chromophore.Derivatization with DCC at 37 °C for 90 min in a basicsolution, with loss of HCl, generates a product thatabsorbs light at 254 nm. Separation of the desired productfrom excess derivatizing agent is possible in 10 min by CEwith a detection limit of 1 ng L�1..265/

Rimantadine is a synthetic analog of amantadine. Bothare antiviral agents used for prophylaxis and treatment ofinfluenza A. Because rimantadine is almost transparent inthe UV/VIS range, either indirect detection or derivatiza-tion has to be used to detect this compound. The indirectdetection method used 5 mM 4-methylbenzylamine in1 : 4 methanol–water as absorbing background electrolytefor detection at 210 nm. The derivatization method usedrimantadine to react with 1,2-naphthoquinone-4-sulfonicacid in alkaline medium. CE determination of the deriva-tive at 280 nm was performed in an uncoated capillary(44 cmð 75 µm ID) using a 40 mM tetraborate buffer atpH 9.2. The detection limits were 0.1 and 2 mg L�1 forindirect detection and derivatization methods, respec-tively. The methods were used to determine rimantadinein pharmaceutical products and for dissolution testing ofFlumadin tablets..266/

Most of the naturally occurring amino acids do not haveproper UV/VIS chromophores for CE analysis. Derivati-zation is a necessity for the determination of amino acidsat reasonable concentrations. By using dansyl chlorideto derivatize the amino acids, the enantiomeric formsof novel depsipeptide antitumor antibiotic BMY-45012,and its analogs, were determined by CE with UV/VISdetection..267/ The compounds were subjected to totalhydrolysis in a vacuum hydrolysis tube with 6 M HCl at110 °C for 24 h. The hydrolyzed residue (about 7.8 mg)was dissolved in water–acetonitrile solvent. For subse-quent derivatization, the solution was further diluted toabout 1.7 mg mL�1 with water. Dansyl chloride dissolvedin acetonitrile (3.0 mg mL�1) was utilized for the deriva-tization of standard native amino acids and those presentin the hydrolyzate. A 50-µL sample solution was mixedwith 50 µL of dansyl chloride solution and an aliquot of



borate buffer at pH 9.08. The mixture was held at roomtemperature for 2 h and used directly for injection.

The aminocychitol antibiotic amikacin can be deriva-tized with 1-methoxycarbonylindolizine-3,5-dicarbalde-hyde at room temperature for 15 min before determi-nation in plasma by micellar CE with UV detection at280 nm and standard fluorescence detection (excitationat 414 nm, emission at 482 nm)..268/ However, there wasonly a twofold gain in sensitivity on switching from UV tofluorescence detection of the amikacin derivative. If laserexcitation can be used (as discussed in the next section),the gain in sensitivity should be significantly higher.

4.2 Derivatization for Fluorescence Detection

In principle, almost any detection mode can be combinedwith a derivatization procedure. In practice, fluorescencemonitoring is favored in most cases. The principles of flu-orescence and the prerequisites for a good fluorophore,including the potential of using diode lasers in combi-nation with a labeling procedure, have been reviewedpreviously..261/

LIF detection is now commercially available for CE,and more and more industrial scientists are using LIFdetection to detect trace amounts of analytes in complexmatrices. LIF detection has several advantages. First, itoffers excellent sensitivity; under optimized conditions,a level of 10�10 M is feasible..269/ This is critical topharmaceutical applications because sensitivity is oftenthe most important parameter to consider when analyzingbiological samples. Second, LIF offers a certain selectivitybecause, unlike UV absorbance, LIF selectively detectscompounds that are fluorescent at certain wavelengths,and most organic compounds do not fluoresce. On theother hand, the fact that most organic compounds do notfluoresce is also a major limitation of LIF detection.Since the available excitation wavelengths of variouslasers are limited, derivatization is often required whenusing CE with LIF detection. The following examplesdemonstrate the applicability of derivatization in CEwith LIF detection for pharmaceutical analysis.

Captopril, an antihypertensive agent, was derivatizedthrough the thiol group with a dicarbocyamine labelto give a fluorescent derivative with a wavelengthmaximum at 675 nm..270/ This wavelength was compatiblewith a semiconductor laser at 670 nm, which was usedfor LIF detection in conjunction with CE. Using amethanol–borate running electrolyte, CE also separatedother labeled thiols such as cysteine and reducedglutathione from derivatized captopril (Figure 15). Thedetection limit of 2.5ð 10�8 M for captopril is limited bydilution due to the derivatization reaction.

Amines can easily be derivatized with FITC isomerI and analyzed by CE using alkaline buffers with or

N

CH3C CH2

(CH2)4SO3

−

N

C

O

IXCH3

H3C

CH3

CY5.3a.IA: X = −NH−

N

COHO

O

HSCH3H

••

Captopril

+

40 30 20 10 0

Migration time (min)

Rel

ativ

e pe

ak h

eigh

t

7

5

2 1

6 43

Figure 15 Electropherogram of a number of thiols labeledwith CY5.3a.IA: (1) unreacted label; (2) captopril; (3) DL-homo-cysteine; (4) L-cysteine; (5) 3-mercaptopropionic acid; (6) 2-mer-captoacetic acid; (7) reduced glutathione. [Reproduced withpermission from Couderc et al..264/]

without dodecyl sulfate micelles. Ramseier et al. reportedthe determination of FITC-derivatized amphetamine,methamphetamine, 3,4-methylenedioxymethamphet-amine and b-phenylethylamine in human urine by CEusing chip-based and fused-silica capillary instrumenta-tion with LIF detection..271/ The results obtained viadirect labeling of fortified urine were compared withthose generated after FITC labeling of urinary extractsthat were prepared by SPE. Using 5 mL of urine with a‘spiked amine’ to FITC ratio of 1 : 250, the SPE extracthad a sensitivity of 200 ng mL�1 urine. That value is rel-evant for toxicology drug screening and confirmation.In contrast, with direct labeling of 10 µL of urine thathad been alkalinized and diluted for derivatization, thelimit of identification was 10 µg mL�1, a value that is toohigh for practical purposes. Compared with fused-silicacapillaries, electrophoresis in microstructures is shown toprovide faster separation and higher efficiencies withoutloss of accuracy and precision.



4.3 Special Applications

4.3.1 Chiral Confirmation

With proper derivatization, CE has been used for thechiral separation of enantiomeric forms of derivatizedamino acids. Liu et al. reported the determination ofenantiomeric forms of amino acids derived from thenovel depsipeptide antitumor antibiotic BMY-45012,and its analogs, the proposed structure of which isshown as (1) [reproduced with permission from Liuet al..267/]. Amino acids were analyzed by completehydrolysis and the hydrolyzate was derivatized with eitherdansyl chloride for UV absorbance detection or FITCfor LIF detection in CE. For fluorescence detection,the fluorogenic reagent FITC was dissolved in acetone(0.01 M) as a stock solution. Amino acids were derivatizedwith FITC-derivatizing solution (5.0ð 10�4 M) underbasic conditions (borate buffer, pH 9.08). The reactionwas allowed to proceed for 2–4 h in the dark atroom temperature and then stored at �20 °C prior touse. Both a metal chelate chiral CE method and acyclodextrin-mediated host–guest interaction approachin micellar electrokinetic chromatography (MEKC) withLIF detection confirmed the presence of several chiralamino acids, such as D-serine and L-b-hydroxyl-N-methylvaline, and the nonchiral amino acid sarcosinein the proposed structure. These methodologies providea quick and sensitive approach for the determinationof amino acid racemization in pharmaceutical naturalproducts and have proven to be useful for structuralelucidation refinement.

4.3.2 Oligosaccharide Determination

The derivatization of the pseudo-oligosaccharideacarbose (2) and its main metabolite (3) with

7-aminonaphthalene-1,3-disulfonic acid (ANDS)(Scheme 1) in human urine allowed the on-column LIFdetection of the pseudo-oligosaccharides in human urinein the nanomolar range..272/ Before derivatization, 0.5-mL samples of urine or spiked urine were evaporatedto dryness. A 100-µL volume of 0.08 M ANDS solutionin acetic acid–water (3 : 17 v/v) and a 50-µL volume of0.9 M NaCNBH3 solution in dimethyl sulfoxide (DMSO)were added to each sample residue. The reagent solutionswere freshly prepared before derivatization. The reactiontubes were vortex mixed and then incubated overnightat 40 °C. The efficient separation of these derivatives byCE using 100 mM triethylammonium phosphate buffer atpH 1.5 allowed the quantitation of acarbose and (3) inhuman urine after application of 300 mg of acarbose.

4.3.3 Derivatization of Protein Samples

With the increased interest in the therapeutic use of therecombinant monoclonal antibody (rMAb) technique, ageneric analytical approach for the analysis of size-basedrMAb variants is desired. CE/LIF with proper derivatiza-tion has been shown to be promising as a general method.The rMAb can be derivatized with a neutral fluorophore,e.g. 5-carboxytetramethylrhodamine succinimidyl ester(5-TAMRSE) for CE/LIF analysis..273/ Samples contain-ing 2.5 mg of rMAb were buffer exchanged into 800 µL of0.1 M sodium hydrogencarbonate (pH 8.3) using a NAP-5column. A 10-µL volume of 5-TAMRSE (1.4 mg mL�1)dissolved in DMSO was then added to 190 µL of rMAbsolution and the resultant mixture incubated for 2 h at30 °C. After incubation, 190 µL of the antibody–dye con-jugate was loaded on to a second NAP-5 column andcollected in 700 µL of 0.1 M sodium hydrogencarbon-ate (pH 8.3). The labeled sample, after incubating with

N

NCH3O

O

NH

NN OR1

O

CH3 CH3

NH3C

O

HON

O

CH3O

N O

R2O

O

H

NN

N

N

CH3

NO

N

O

O

HN

N

OCH3

O

HO

O

O CH3

CH3

OHCH3

OO

(1)

Compound R1 R2

BMY45012 CH(CH3)2 CH(CH3)2

Analog-G439B CH2CH3 CH2CH3

Analog-G451 C(CH3)3 C(CH3)3

Analog-G435 C(CH3)3 CH(CH3)2

Analog-G439A CH(CH3)2 CH3



OCH2OH

HOOHHO

O

OCH2OH

HOHOO

OCH3

HOHO

HN

HO

HOHO

CH2OH

(2)

OCH2OH

HOHOO

OCH3

HOHO

HN

HO

HOHO

CH2OH

(3)

OH

OCH2OH

HOHOO

OH

Carbohydrate

COH

CH2OH

HOHOO

OH

SO3H

SO3HH2N

COH

CH2OH

HOHOO

NH

SO3H

SO3H

Schiff base

NaCNCH3

CH2

OHCH2OH

HOHOO

NHSO3H

SO3H

Scheme 1 Reaction of (2) and (3) for derivatization withANDS by reductive amination. [Reproduced with permissionfrom Rethfeld and Blaschke..272/]

SDS, can be separated by CE using a hydrophilic poly-mer as a sieving matrix. Using these precolumn labelingconditions, the detection of rMAb at a low-nanomolarconcentration (9 ng mL�1) is obtained with no apparentdecrease in resolution or changes to the distribution ofrMAb analyte species in comparison with an unlabeledsample. This assay can be used in monitoring consistency

of the bulk manufacture of a protein pharmaceutical andin providing a size-based separation of product-relatedvariants and also nonproduct impurities. The CE/LIF withderivatization demonstrated comparable resolution andsensitivity to silver-stained sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS/PAGE) but offeredthe advantages of enhanced precision and robustness,speed, ease of use, and on-line detection.

CE was used for the study of palmitoyl derivatizationof interferon-a2b (p-IFN-a)..274/ The derivative wasprepared by covalent attachment of the fatty acid tolysine residues in the protein through a reaction with N-hydroxysuccinimide palmitate ester (Scheme 2). The firststep involved the preparation of an intermediate (NHSP),which in turn was reacted with IFN-a. CE was able tostudy the effect of reaction time and reagent/protein ratio.

4.4 Derivatization Modes

In general, derivatization in CE can be accomplished byeither pre- or postcolumn derivatization. The precolumnderivatization is similar to methods used for derivatizationin HPLC. Since CE has a much superior separation power,the interference from the derivatization by-products maybe less of a problem than in LC. However, CE is verysensitive to the ionic strength of the sample. Additionalsample preparation may be needed if the mixture in thederivatization reaction has a high ionic strength.

R COOH NHO

O

O

+ DCCNO

O

O

R + DCU

Active ester (isolated)

NH2 IFNpH 7.2

+α

R C NO

HIFN α

Scheme 2 Synthetic process for the fatty acrylation of IFN-a.R�COOH palmitic acid. [Reproduced with permission fromFoldvari et al..274/]

Postcolumn detection strategies have also been devel-oped for CE. An overview of the advantages and limita-tions of postcolumn derivatization for CE can be foundin a recent paper..261/ The details about the instrumentaldevelopments and applications of post-column derivatiza-tion in CE can also be found in the literature..262/ Varioussystems to merge the reagent solution with the separationmedium have been developed, including coaxial capillaryreactors, gap reactors, and free-solution or end-columnsystems. For all reactor types, the geometry of the system,



and the method used to propel the reaction mixture (bypressure or by voltage) appear to be critical to preservethe separation efficiency. To minimize peak broadening,careful design in terms of connecting a reagent capillary toform a tee is necessary. Because of the small (50–75 µm)ID of the capillaries used, normal mixing through a dif-fusion process is sufficient. The most frequently appliedreactors are (1) co-axial, (2) gap, (3) free-solution, and(4) sheath flow. With proper design, plate numbers ofover 100 000 could be realized. The strict requirementson the reaction rate in postcolumn derivatization in CElimit the number of different reagents that have beenused. For LIF detection, mainly OPA and its naphtha-lene analog have been used. With careful instrumentdesign, the detection limits of postcolumn derivatizationcan be comparable to those of precolumn derivatization.

5 CONCLUSION

It can be argued that the advances made in MSdetection for GC, HPLC, and CE have diminishedthe importance of chemical derivatization for thesetechniques. However, chemical derivatization of drugsis critical for GC because these samples, which oftencontain multiple polar substituents, are simply not volatileor thermally stable. MS detection for HPLC and CEis still expensive and requires considerable operatorexpertise. Chemical derivatization with HPLC to permitfluorescence detection is certainly one of the moredesirable methods for routine use to solve selectivity anddetectability problems for drug samples not amenable toGC. As the cost of laser technology comes down, thesame statement will eventually be true for CE also.

ABBREVIATIONS AND ACRONYMS

ANDS 7-Aminonaphthalene-1,3-disulfonicAcid

BSA N,O-Bis(trimethylsilyl)acetamideBSTFA N,O-Bis(trimethylsilyl)trifluoro-

acetamideCBD CannabidiolCE Capillary ElectrophoresisDCC DiethyldithiocarbamateDES DiethylstilbestrolDMES DimethylethylsilylDMFDA N,N-Dimethylformamide

DimethylacetalDMIPS DimethylisopropylsilylDMSO Dimethyl SulfoxideEC Electron Capture

FFAP Free Fatty Acid PhaseFITC Fluorescein IsothiocyanateFMOC 9-Fluorenylmethyl ChloroformateGC Gas ChromatographyGC/MS Gas Chromatography/Mass

SpectrometryHFAA Heptafluorobutyric AnhydrideHFBI HeptafluorobutylimidazoleHMDS HexamethyldisilazaneHPLC High-performance

Liquid ChromatographyLC Liquid ChromatographyLIF Laser-induced FluorescenceMBTFA N-MethylbistrifluoroacetamideMEKC Micellar Electrokinetic

ChromatographyMS Mass SpectrometryMSTFA N-Methyl-N-trimethylsilyl-

trifluoroacetamideMTBSTFA N-Methyl-N-(t-butyldimethylsilyl)-

trifluoroacetamideOPA o-PhthalaldehydePFAA Pentafluoropropionic AnhydridePFAI PentafluoropropionylimidazolePFB-Br Pentafluorobenzyl BromideQUAT Quaternary Ammonium SaltrMAb Recombinant Monoclonal

AntibodySDS Sodium Dodecyl SulfateSDS/PAGE Sodium Dodecyl Sulfate

Polyacrylamide Gel ElectrophoresisSPE Solid-phase ExtractionTBDMS t-ButyldimethylsilylTFAA Trifluoroacetic AnhydrideTFAI TrifluoroacetylimidazoleTMAH Trimethylanilinium HydroxideTMCS TrimethylchlorosilaneTMS TrimethylsilylTMSI N-TrimethylsilylimidazoleUV UltravioletUV/VIS Ultraviolet/Visible5-TAMRSE 5-Carboxytetramethylrhodamine

Succinimidyl Ester

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Pharmaceuticals and Drugs (Volume 8)Eluent Additives and the Optimization of High-performance Liquid Chromatography Procedures ž Gasand Liquid Chromatography, Column Selection for, inDrug Analysis ž Planar Chromatography in Pharmaceu-tical Analysis ž Solid-phase Extraction and Clean-upProcedures in Pharmaceutical Analysis



Gas Chromatography (Volume 12)Gas Chromatography: Introduction ž Column Technol-ogy in Gas Chromatography ž Sample Preparation forGas Chromatography

Liquid Chromatography (Volume 13)Liquid Chromatography: Introduction ž Capillary Elec-trophoresis ž Column Theory and Resolution in LiquidChromatography ž Normal-phase Liquid Chromatogra-phy ž Reversed Phase Liquid Chromatography

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