hplc general

Jordan Journal of Pharmaceutical Sciences, Volume 1, No. 1, 2008

Current HPLC Methods for Determination of Medicaments inFormulations and Biological Samples

1 2 1 Mitsuhiro Wada , Suleiman M. Alkhalil and Kenichiro Nakashima

1Department of Clinical Pharmacy, Graduate School of Biomedical Sciences, Nagasaki University,Nagasaki, Japan.

2Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, University ofJordan, Amman, Jordan.

ABSTRACT

The performance of high-Performance liquid chromatography (HPLC) instruments has been remarkably

progressed. As a result, an HPLC method plays a conspicuous role in analysis of medicaments in formulations

and biological samples. The varied detection methods used for HPLC such as ultra violet (UV), mass

spectrometry (MS), fluorescence (FL) and chemiluminescence (CL) detections in addition to electrochemical

detection (ECD) with suitable pretreatment or labeling were chosen in response to the purpose of analysis.

Analysis of medicaments in formulations was mainly performed by UV detection, meanwhile EC, MS, FL and

CL detections with high sensitivity were used for analysis of medicaments in biological samples. The sensitivity

ranging from microgram to picogram level could be achieved. In this review, current HPLC methods for

determination of medicaments in formulations and biological samples were described. Furthermore, their

advanced applications for chiral analysis and pharmacokinetic drug-drug interaction evaluation of medicaments

were presented.

Keywords: HPLC, Medicaments, Formulations and Biological Samples.

INTRODUCTION medicaments. In clinical practice, immunoassays which

offer appropriate sensitivity for the detection of

Analysis of medicaments in formulations or medicaments in biological specimens are utilized such as(2)

biological fluids is required to promote for rational use fluorescence (FL) polarization immunoassay ,(3)

of medicament. Any useful information for quality chemiluminescence (CL) immunoassay and enzyme-(4)

control, pharmacokinetics, pharmacodynamics, linked immunosorbent assay . Exclusive kits andpharmacology and toxicology of medicament can not be automated instruments for each medicament have been

obtained without it. Especially, therapeutic drug developed and widely spread. However, the cross

monitoring based on determination of medicaments is reaction sometimes makes overestimation for the(5)

one of the requisite factors for performing appropriate determination of each component in the samples .(1)

treatment by some medicaments . On the other hand, a separation technique is requiredVaried methods have been used to analyze in the cases as follows:

1. Simultaneous determination of coadministratedReceived on 5/9/2003 and Accepted for Publicat ion on medicaments such as anticonvulsants.12/9/2007. 2. Simultaneous determination of parent compound

E-mail: [email protected]

- 1 - © 2008 DAR Publishers/University of Jordan. All Rights Reserved.

Current HPLC… Mitsuhiro Wada et al.

and active metabolites. detection method in the aims of analysis, e.g., sensitive,

3. Analysis of racemic compounds. selective, rapid, simple or inexpensive determinations.

Among separat ion techniques employed for The aim of this review is to overview current HPLC

medicament analysis such as thin-layer methods for determinat ion of medicaments in(6)

chromatography , gas chromatography (GC), high- formulations and biological samples. The varied(7)

performance liquid chromatography (HPLC) , capillary detection methods of HPLC such as UV, MS, FL, CL(8) (9)

electrophoresis and capillary electrochromatography , and ECD will be mentioned. In each following section,GC and HPLC have proven to be the most popular, representative interesting results including our recent

because these can offer better sensitivity, selectivity and publications were presented. Furthermore, their

applicability. Especially, HPLC shows excellent advanced applications for chiral analysis or

capability for the analysis of aqueous samples. Most pharmacokinetic drug-drug interaction evaluation of

medicaments as well as endogenous components in medicaments will be mentioned.

biological samples are commonly non-volatile polar

compounds, and thus HPLC is more suitable than GC for HPLC Methods for Determination of Medicaments in

their analysis. Formulations

One of the major advantages of HPLC for analysis of

medicaments is the improvement of stationary phase. Medicaments in formulations

The separation of medicaments with more than several Numerous HPLC methods for determination of

ten thousands theoretical plates/m can be performed. medicaments in formulations have been reported. These

Though a conventional reversed-phase ODS (C18) methods have been applied to determine medicaments in(23,32)

column is the most frequently used for this purpose, various formulations such as tablet, capsule ,(10) (11-16) (33)

other kinds of stationary phases such as C4 , C8 , dermatological formulation , multi-component(17) (18-20) (34) (10)

C30 and NH2 columns are also chosen according syrup and aerosol . In general, since formulationsto a characteristic of analyte. A normal-phase silica contain relatively large amounts of medicaments ranging

(21)column is also used for analysis of medicaments or from sub-microgram to several hundred

(22)those with labeling due to their hydrophobic milligram/formulation, highly sensitive determination isproperties. Recently, a monolithic column has attracted not always necessary. On the other hand, a strict-

attention as an alternative option for HPLC. Since validated analytical method is required. The objective of

monolithic support plays as continuous homogenous validation of an analytical procedure is to demonstrate

phases, users can perform chromatography with much whether it is adequate or not for its intended purpose. A

lower pressure than that of conventional packed column. number of parameters must be investigated in order to

Therefore, much faster separations at high flow rates can validate the analytical methods, e.g., precision, accuracy,

be achieved with high efficiency and reduction of specificity, limits of quantification (LOQ) and detection(35)

running time. Separations of E-lactamic antibiotics (e.g., (LOD), linearity and robustness defined by ICH .(23) (24)

amoxicillin, ampicillin and cephalexin) , and sotalol Precision is measured as repeatability, intermediatewere performed with a monolithic column. Furthermore, precision and reproducibility. The repeatability (intra-

a specific column for chiral separation of medicaments day) and intermediate (inter-day) precision of the(21, 25-31)

can be available . method are demonstrated by analyzing the sampleAnother advantage of HPLC is a variety of detection reference standard solution of known concentration

methods including ultra violet (UV), mass spectrometry during 1 day and each of several days under the same

(MS), FL and CL detections in addition to electrochemical conditions. Reproducibility refers to use of the analytical

detection (ECD). An operator can select the suitable procedure in different laboratories. Accuracy is

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de te rmined by ana lyz ing a sample o f known Medicaments in biological samples

concentration of standard spiked in sample and The varied matrices such as urine, blood (whole blood,

comparing the measured value with the true value. serum and plasma), tissue homogenate and dialysate were

Specificity of method is determined by a placebo used for biological analysis of medicaments. Commonly

analysis. Placeboes containing all additives except pretreatment of biological samples is required before

ingredient are prepared for this study. They are treated in injection into an HPLC system. The simplest pretreatment

the same manner as the normal samples. To compare the is deproteinization with denaturants such as organic

chromatogram of sample with that of placebo, the specificity solvents or acids. After deproteinization, the mixture is

of the method could be confirmed. The LOQ and LOD are centrifuged and the resultant supernatant is subjected to

calculated as correspondence concentrations with a signal-to- analysis. A deproteinization method is advantageous as it

noise (S/N) ratio of 3 and 10, respectively. In addition to this, is convenient and more economical compared to a liquid-

the LOD (or LOQ) is expressed in equation (1), liquid extraction and solid-phase extraction (SPE)

methods.

LOD (or LOQ) =3.3 (or 10) ı/s (1) Liquid-liquid extraction is another alternative for

sample clean-up. Water-immiscible organic solvents are

where ı is the standard deviation of the response and used for extraction of analytes from biological fluids. SPE

s is the slope of the calibration curve, and defined as a has become more popular for analyses of medicaments in

five (or three) times of standard deviation of the biological fluids due to the excellent figures such as

appropriate blank response. The LOQ was defined as selective retention of the analytes, reduction of sample

the concentration which gives less than 20 % of RSD size and solvent requirements, high throughput and the

and/or less than ±20 % of accuracy. Linearity should possibility of automation. Recently, liquid-phase (LPME)

be evaluated by an appropriate statistical method such and solid-phase microextraction (SPME) are known as

as least squares method. For establ ishment of new and effective sample preparation techniques. In

l i nea r i t y , a m in imum o f 5 concen t r a t i ons i s LPME, medicaments can be extracted from aqueous

recommended by ICH. The robustness of the method biological samples, through a thin layer of organic solvent

is a measure of its capacity to remain unaffected by immobilized within pores of the wall of porous hollow(48, 49)

small , but deliberate, variations in the method fiber . For SPME, fibers and capillary tubes coatedparameters such as pH value of mobile phase, and with an appropriate stationary phase are usually used.

provides an indication of its reliability during normal Moreover, microextraction in a packed syringe and stir-

usage. Heyden et al . reviewed for robustness in bar-sorptive extraction using coated magnetic stir bar have(36) (50)

method validation . been developed . In both techniques, microliter level ofA part of current HPLC methods for determination solvent was required, and thus the solvent consumption in

of medicaments in formulations is summarized in pretreatment could be minimized.

Table 1. Since matrix of formulat ion is not so Ultrafiltration, a straightforward sample preparation

complicated, time-consuming pretreatment and a method, is also utilized as a convenient and rapid

compl ica ted e lu t ion sys tem for separa t ion of pretreatment of biological fluids to avoid tedious

medicaments are not required. Almost medicaments extraction and evaporation techniques. Aliquots of

are extracted by ultrasonication. The most methods plasma or serum sample are simply fi l tered by

listed in Table 1 utilized UV detection. An HPLC-UV centrifugation with a micron filter, and the resultant is

method is simple and practical for determination of a injected onto an HPLC system.

medicament having a chromophore with absorption A direct injection method is a preferred technique for

bands in the UV or visible region. the analysis of biological fluids concerning time- and

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cost-consumption, analyte loss and simplicity. Direct medicaments by UV detection depends on their

injection is generally combined with column switching. absorptivity, and is generally inferior to those of MS, FL

The column switching technique allows the adsorption or ECD. However, an HPLC-UV method is precise and

of analyte on a pre-column and removal of the frequently used for quality control of medicaments in

interfering components such as protein, followed by formulations. Because medicaments contained in

back-flushing and transfer of the analyte to an analytical formulations are relatively in large amounts, precise(51)

column by switching the direction of the effluents . ident i f icat ion and quant i f icat ion of the act iveMicrodialysis is often utilized as a sampling tool to components and the impurities are important for the

monitor concentrations of free-form drugs or safety and efficacy of formulations. The impurities and

neurotransmitters, and is also applied to potential degradation products can be present in bulk and

neuropharmacological and pharmacokinetic studies. A formulations and change the chemical, pharmacological

microdialysis probe consists of a small semipermeable and toxicological properties. Rao et al. reported a rapid(38)

membrane, on which analytes are recovered by passive and simple HPLC-UV method for paracetamol .diffusion (Fig. 1). As the advantages of microdialysis, long- Paracetamol, nine impurities and one degradation

term sampling is possible with minimal damage for organs product in the formulation were successfully determined

or tissues and the clean-up procedure is not ordinarily within 50 min. Stabi l i ty- indicat ing assays for(52) (37) (39)

required . Many application studies on pharmacokinetics SK3530 , ofloxacin , donepezil (DP)(44) (82)

and drug-drug interactions using microdialysis have been hydrochloride and phenylpropanolamine (PPA)(17, 53, 54)

reported . in bulk and formulations using HPLC-UV detectionA highly sensitive analytical method determining were also reported. In our previous report, PPA,

less than sub-nanogram level is generally required for caffeine and chlorpheniramine in commercially

analyses of medicaments in biological samples. In available over-the-counter preparation were well

pharmacokinetic studies, low concentration levels of separated (Fig. 2) and simultaneously quantified

medicaments in samples should be determined for wi th the l imi t of de tec t ion of sub-mi l l imolar(45)

several hours after administration. Sensitivity of the levels .analytical method is required according to the following On the other hand, since the performance of UV

conditions; detector has been recently improved, theDose of medicaments. determination of medicaments in biological samplesStability of medicament in living body. was a l so ach i eved by an HPLC-UV me thod .

Amount of matrix obtained. Levomepromaz ine , c lozap ine and the i r main(11)

For this purpose, HPLC-MS (or -MS/MS), -FL, -CL metabolites in human plasma , aspirin in human(59) (83)

or -ECD were mainly used (Table 2). Characteristics of serum , docetaxel and paclitaxel in plasma , DP(27, 84)

these methods will be mentioned in the next paragraph. in plasma , fluoroquinolones such as enoxacin,(15)

In some cases, a labeling reaction was combined with ofloxacin and norfloxacin in chicken blood , non-steroidal(28)

these methods as the occasional demands. Labeling can anti-inflammatory drugs, ketoprofen, meloxicam ,(29) (60)

serve improve the sensitivity, selectivity and flurbiprofen , ibuprofen and dicrofenac sodium and(53)

chromatographic behaviors. triazolam in plasma were determined. An HPLC-UVdetection was also applied to pharmacokinetic studies of

(55) (56) (14)HPLC-UV detection tenatoprazole , ferulate sodium , clopidogrel

(58,85,86)The advantages of HPLC-UV detection are wide and paclitaxel . However, relatively large

adaptablity and simplicity using a relatively inexpensive amounts o f b lood sample (200-1000μl ) were

ins t rument . The sensi t iv i ty of the analys is of consumed for determination of medicaments.

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HPLC-ECD detection drug efficacy and toxicity. HPLC-MS is a powerful

The ECD detection is accepted as a sensitive and analytical tool that can facilitate the measurement of(89)

selective technique for the determination of electroactive medicaments and biomarkers . Compared with GC-substances and has been used for quality control of MS, HPLC-MS is well-suited for sensitive quantification

(25)medicaments. Levodopa methyl ester and tanshinone of polar and non-volatile medicaments and has become a

(87)IIA in formulations containing electroactive widely applied technique in recent years. MS detectionfunctional groups such as phenol were detected with provides both qualititative and quantitative information

LOD of a few nanogram per milliliter. Nakao et al. for each analyte. HPLC-UV, -FL and -ECD rely on the

reported an HPLC-ECD detection method for quality retention time of the analytes, whereas MS can identify

control of positron emission tomography (PET) the analytes even in the presence of co-eluted impurities(12)

radiopharmaceuticals . In 19 PET pharmaceuticals by us ing a s ing le ion mon i to r ing mode . Thestudied involving methionine, dopa, methylspiperone development of interfaces between HPLC and MS was

and verapamil, these compounds and their corresponding very important to build up the valuable HPLC-MS

precursors used in the synthesis of the system. An interface is devised to separate small

radiopharmaceuticals were quantified. This method could be amounts of analytes from large volumes of solvents and11

applied to the analysis of [ C] MP4A, a useful PET only can ionize analytes. Two ionization modes, anradiopharmaceutical having no available UV absorbance for atmospheric pressure chemical ionization (APCI) and an

measuring acetylcholinesterase activity in the brain. electrospray ionization are mainly used for the

Moreover, determination of medicaments in interfacing of MS for the measurement of medicaments.

biological samples, e.g., urine, plasma, serum and tissue, The development of these ionization sources has enabled

by HPLC-ECD was demonstrated with suitable the evolution of this technique to the point where HPLC-(7)

pretreatment of the sample such as liquid-liquid MS is routinely used for pharmacokinetic studies . Due(30,61-64,87,88)

extraction or SPE . However, the ECD is to its high sensitivity and selectivity, an HPLC-MShardly compatible with gradient elution. This fact is the method is mainly used for quantification of medicaments

main disadvantage for ECD, and generally limits its use and biomarkers in biological samples. As shown in Table 2, a

to the sole separation of a couple of medicaments, e.g., number of methods hase been established for determining of(66)

metoclopramide, imipramine, diclofenac and medicaments such as triptolide, wilforide A, triptonide ,(87) (67) (68) (69)

hydrochlorothiazide, in a single run . Therefore, the indapamide , cyclovirobuxine , gambogic acid and(90)

analysis of single medicament by ECD has been dexamethasone .(62)

extensively performed for clenbuterol , 5- Tandem MS (-MS/MS) is a tool in which two mass(63) (64)

hydroxyoxindole and vitamin K . However, using a new spectrometers are connected by a fragment chamber.generation coulometric detector, simultaneous determination Compared to HPLC-MS, more sensitive and selective

of ten commercially available macrolide antibiotics such as determination could be accomplished with tandem

erythromycin, dirithromycin, tylosin, tilmicosin, spiramycin, MS/MS. MS/MS detect ion can dis t inguish the

josamycin, kitasamycin, rosamicin, roxithromycin and compounds which have the same intact mass and

oleandomycin in human urine was achieved with LOD of 2.5 provide additional information enhancing the reliability(61)

ng on column . of the method. Applying HPLC-MS/MS detection hasled to the development of rapid and sensitive methods

HPLC-MS (/MS) detection for analyses of many kinds of medicaments such as(20) (31) (16)

Measurement of small amounts of medicaments and glucosamine , warfarin , cetirizine ,(91) (92)

pharmacodynaminc biomakers in biological samples rosiglitazone , neomycin and bacitracin in bodyprovides the opportunity for a better understanding of fluids. Vainchtein et al. developed an HPLC-MS/MS for

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paclitaxel and its 3’-p- and 6-Į-hydroxy metabolites in amounts of plasma because of their intense FL.

human plasma with a simple liquid-liquid extraction However, in most cases, the intrinsic FL intensity of the(65)

procedure . Paclitaxel and its metabolites were rapidly medicaments is not strong and co-existing compounds inseparated within 9 minutes of run time and sub- matrices interfere its determination.

nanogram per mil l i l i ter of substrates could be If compounds do not have fluorescent properties

determined. Although MS/MS detection is very described above, a labeling reaction is recommended in

sensitive, it is too expensive to use for the routine order to introduce such a characteristic in the molecule.

measurements in the pre-clinical laboratory. A labeling reaction is commonly achieved by the

selective reaction between the functional group of theHPLC-FL detection analyte and that of the labeling reagent. Labeling serves

An HPLC-FL detection method has been extensively to improve the sensi t ivi ty , select ivi ty and the

employed for the determination of trace amounts of chromatographic behavior. Many labeling reagents have

compounds and favorably comparable to UV detection been developed for each reactive functional group.

in regard to sensitivity and selectivity, because FL Representative fluorescent labeling reagents are shown

detection counts the increase in photon number occured, in Fig. 4. To date, several reviews describing the(95-98)

while UV detection measures the ratio of transmitted labeling reagents for HPLC have been published .(46) (18) (26)

light that passes out through the sample solution and the Amantadine , voglibose and fluoxetine inoriginal irradiation light. Additionally, for FL detection, formulation were determined by an HPLC-FL method

two wavelengths for excitation and emission should be with FL labeling. Sensitive and reliable methods with 9-

selectively used; this is the major reason why FL fluorenylmethyl chloroformate (FMOC, Fig. 3-c) for(99)

detection is more selective than UV detection. determination of vertilmicin and alendronate(78)

Furthermore, its instruments are inexpensive compared sodium were established. 4-(4,5-Diphenyl-1H-with MS (or MS/MS) detection. imidazol-2-yl) benzoyl chloride (DIB-Cl, Fig. 3-d) was

Medicaments with their intrinsic FL can be detected one of the most powerful fluorescent labeling reagents

by HPLC-FL without pretreatment such as labeling. For and has been appl ied to de te rmine the var ied(76)

instance, recombinant human erythropoietin in medicaments such as mazindol and its metabolite ,(54, 100) (101)

pharmaceutical products was determined by anion- PPA and atomoxetine . Especially, the method(93)

exchange chromatography . P ro te in b ind ing o f for PPA showed applicability to the varied biologicalnaproxen based on measurement of its intrinsic FL was samples such as human plasma, rat brain and blood

(48)studied . In biological samples (mainly plasma), microdialysates. The proposed method showed high

(73) (72)analyses of nabumetone and its metabolites , sensitivity with LOD of 0.14 ng/ml in plasma using only

(74) (75)camptothecin and doxazosin were performed. In 100 μl of sample, and 2.4 and 2.9 ng/ml in brain andour previous report, by utilizing intrinsic FL of DP blood microdialysates, respectively, using 15 μl of

hydrochloride, rapid and sensitive determination of DP microdialysates. This method could be useful for PPA

in human plasma, rat plasma and microdialysis was monitoring in human plasma for clinical and forensic

achieved with a short C30 column by an isocratic purposes.(19)

elution . As shown in Fig. 3, DP was separated within However, FL determination of medicament having15 minutes and LOD of DP in human plasma, rat plasma no functional group is difficult, because it can not be

and microdialysis ranged from 0.2-2.1 ng/ml (S/N=3). labeled with a typical fluorescent labeling reagent.

Moreover, pharmacokinetic studies of medicamensts Therefore, instead of mother medicament, its metabolite(94) (24)

such as MCC-555 (thiazolidineones) , sotalol and having a reactive functional group was used for analysis.(76)

atenolol could be achieved by consuming small 4-(4-Bromophenyl)-4-hydroxypiperadine, a bromperidol

- 6 -


metabolite was determined with 4-fluoro-7-nitro-2, 1, 3- (N-chloroformylmethyl-N-methyl)amino-2,1,3-(72) (108)

benzoxadiazole (Fig. 3-a) . Recently, 4-(4,5-diphenyl- benzoxadiazole and estradiol labeled with dansyl1H-imidazol-2-yl) phenylboronic acid (Fig. 3-k), a novel chloride were also sensitively determined in plasma by

fluorescent labeling reagent for aryl halides was HPLC-PO-CL. Recently, unique HPLC-PO-CL methods(22)

developed . This labeling based on Suzuki coupling combined with UV irradiation were developed for thereaction that is a palladium-catalyzed cross-coupling determination of medicaments, which were converted to

reaction of aryl halides with arylboronic acids. The hydrogen peroxide and fluorescent products. Selective(109) (79)

proposed method was successfully applied to the determination of quinones and atremisinin werequantification of haloperidol in human serum. achieved. Typical chromatograms of artemisinin in

human serum were shown in Fig. 5.2+ 3+

HPLC-CL detection Ru(bpy)3 is electrochemically oxidized to Ru(bpy)3

CL is an emission of light from luminescing on the electrode surface, then, CL detection occurs during3+

species electronically excited by a chemical reaction oxidization of the various amines by Ru(bpy)3 . ȕ-of a precursor of those species. The CL reaction blockers such as atenolol and metoprolol in human urine

(80)doesn’t require an exciting light source. This permits an were determined by this method . Another ECL whereincrease in the detector’s sensitivity, the attainment of a emission of light from oxidized indomethacin by Mn (III)

(81)large S/N ratio and an inexpensive instrumentation. electrogenerated on electrode surface was found . ByTherefore, the CL detection method is of growing this method, sensitive determination of indomethacin in

importance in pharmaceutical and biomedical analysis urine with a LOD of 8 ng/ml (S/N=3) was achieved(102)

due to its low LOD, and a simple instrumentation . without any pretreatment. Numerous applications using anThe CL reactions including luminol, peroxyoxalate HPLC-CL detection have been reported in proportion to

chemiluminescence (PO-CL) and electrogenerated the increase in chemiluminogenic and CL labeling

chemiluminescence (ECL) such as tris(2,2’- reagents described above.2+

bipyridyl)ruthenium(II) (Ru(bpy)3 ) and Mn(III) havebeen well-known. Several reviews for CL reactions have Advanced Application Research

(96, 102-104)already been published .

Among them, luminol is one of the most important Chiral analysis of medicaments by HPLC

CL reagents for the determination of medicament. It has Analysis of enantiomers is very important to provide

known that oxacillin, an antibiotics prolong, can enhance data for pharmacokinetic studies when the enantiomers

the luminescence from luminol. Using this system, possess different pharmacological activities and/or are

unbound oxacillin in human serum was determined with metabolized at a different ratio.(105)

a simple ultrafiltration pretreatment . Chiral separation of medicaments can be achieved by aIn PO-CL system, oxalates or oxamides react with direct or indirect separation. A review described in details on

hydrogen peroxide to yield intermediate peroxides which chiral separation of medicaments has already been(7)

produce light by energy transfer to a co-existing published . In the direct separation, enantiomers or those(103)

fluorophore . Tsunoda et al. reported a sensitive labeled with an achiral reagent are separated with a chiraldetermination method of catecholamines and 3-O- stationary phase. Recently, this direct separation is mainly

methyl metabolites by fluorescence labeling with used because the varied chiral stationary phases such as(106)

ethylenediamine . T h e p r o p o s e d m e t h o d w a s Pirkle-type, cellulose and amylase types can be selected for(21,27) (29)

successfully applied to determine catecholamines in analytes. Enantiomers of DP and flurbiprofen weremouse plasma by on-line labeling. Additionally, determined by an HPLC-UV method with Chiralcel OD and

(107)penbutolol labeled with 4-dimethylaminosulfonyl-7- Chiralpak AD-RH columns, respectively. R/S-Warfarin

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separated with a Chiralpak AD column was determined ITZ were performed. Furthermore, established HPLC-(31)

by HPLC-ACPI-MS/MS . In the indirect separation, FL detection for PPA labeled with DIB-Cl wasenantiomers labeled with a chiral reagent are separated applied to assess the possibility of pharmacokinetic

on an achiral column (e.g., ODS). The reports using an interaction of PPA with caffeine and (100)

indirect separation are not so much, since quality control of chlorpheniramine . These medications are presentthe chiral reagents for labeling is very difficult. An HPLC- in the same formula. In this study, coadministration

UV detection method for ketoprofen labeled with L- of PPA with caffeine and chlorpheniramine showed(28)

leucinamide was reported by Montoya et al. . They significant alteration on pharmacokinetics of PPA in(54)

evaluated the pharmacokinetic parameters of brain .enantiomers in dog after an oral administration at the

recommended dose. Recent chiral analysis methods of CONCLUSION

medicaments were summarized in Table 3.Study on drug-drug interaction Nowadays, the analysis of medicaments plays

Nowadays, multi-medicament use was increased in important roles in the development of new medicaments,

clinical practice, especially, in the treatment of elder quality control of formulation and evaluation of

patients. Therefore, the drug-drug interaction of therapeutic effect. In this paper, current HPLC methods

medicaments, which are expected to be coadministrated for medicaments in formulations and biological samples

in c l in ica l p rac t i ce , seems to be s ign i f i can t . are the subjects of interest. In the recent decade,

Accumulation of this kind of knowledge may be helpful performance including sensitivity, selectivity and

for rational use of medicament in practice. In this mean rapidity of the methods has been rapidly progressed in

HPLC method is a powerful technique to study on drug- proportionality to the progress of instruments, stationary

drug interaction. phases and labeling reagents. Additional remarkable

A semi-micro column HPLC method for triazolam points of current methods are the reduction of

(TZ) in rat plasma and brain microdialysates was consumpt ion o f t he r eagen t s and was te wi th

developed for drug-drug interaction study of triazolam consideration of our environment. In fact, the techniques(53)

with Itraconazole (ITZ) . As a result, ITZ seriously for miniaturization of the system and shorteninginterfered with pharmacokinetic parameters of TZ when analytical time can contribute to achieve those goals.

single simultaneous administration of TZ with ITZ and Hereafter, HPLC will keep occupying an essential

single administration of TZ after daily pretreatment with position to carry on the analysis of medicaments.

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Perfusate

Dialysate

ż: Endogenous compound

Ɣ: Exogenous compound

Figure 1. A microdialysis probe system

Figure 2. Chromatograms obtained from human plasma (A) and spiked with 25 nM of DP (B)

The detector sensitivity in (A) is two times higher than in (B). Printed from Ref. 17 with permission from Elsevier

Sciences, B.V.

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Figure 3. Chromatogram obtained from the soft capsule preparation

(A) Before spiking, with the concentrations 151, 100 and 3.8 mM of caffeine, PPA and CPA. MPPA concentration is 10

mM. (B) The sample spiked with 200 and 7.6 mM of PPA and CPA, respectively. Printed from Ref. 45 with permission

from John Wiley & Sons, Ltd.

- 17 -


For carboxylic acidsFor amines

CHN2F N(CH32)

NO f) ADAM

NCH2OCOCl

NO2 CH2BrSO2Cl

c) FMOCa) NBD-F

b) DNS-Cl g) Br-MMCCH3

MeO O OMeO N O

N OCOCl MeO

N MeO N COCl N SO N NH2

HMeO

e) DMEQ-COCld) DIB-Cl h) DPS-PZ

F For alcohols For aryl halidesFor thiols

NCON

O NN O N

N B(OH)2

SO2NH2N

SO2Ph Hi) ABD-F

k) DPAj) PSBD-NCO

Figure 4. Representative FL labeling reagents.

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Figure 5. Chromatograms of (A) human serum and (B) human serum spiked with artemisinin

Spiked artemisinin concentration, 40 Pmol/L (11.3 Pg/mL). Conditions, mobile phase, 20 mmol/L imidazole buffer (pH

8.50) and acetonitrile (30:70, v/v); CL reagent, 0.50 mmol/L DNPO and 1.50 Pmol/L TMP in acetonitrile, flow rate of 1.0

mL/min; reaction coil length, 6.0 m. Printed from Ref. 79 with permission from John Wiley & Sons, Ltd.

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ŇŒťƕŒ ŗƒƃœŵ ¾őŒƍŪƃŒ œƒżŒŧŹƍřœƆƍŧƂ

1 2 1œƆƒŬœƂœƈ ƍŧƒŬƈƂƍ ¾ƒƄŤƃŒ ƇœƆƒƄŪ � ŒťŒƍ ƍŧƒƌƒŪřƍƆ

1.ƉŕŗŕƔƅŔ ƓƄ� ŔŪŕŠŕƊ ƓƄŔŪŕŠŕƊ� řŸƈŕŠ řƔƏƔţƅŔƏ� řƔŗųƅŔ ƇƏƆŸƆƅ ŕƔƆŸƅŔ ŚŕŬŔũŧƅŔ řƔƆƄ řƔũƔũŬƅŔ� řƅŧƔŰƅŔ ƇŬƁ

2.ƉŧũƗŔ Ɖŕƈŷ� řƔƊŧũƗŔ� řŸƈŕŠƅŔ řƅŧƔŰƅŔ� řƔƆƄ řƔƊƛŧƔŰƅŔ� ŚŕƔƊƂśƅŔƏ ŚŕƔƊƛŧƔŰƅŔ� ƇŬƁ

ŭº ŤƄƆ

ŘŪ» ƎŠƗŔ ƋŨƍ ŚŗŸƅ ƃƅŨƅ řŠƔśƊƏ (HPLC) ŉŔŧƗŔ řƔƅŕŷ ¿œŔƏŬƅŔ ŕƔžŔũŻƏśŕƈƏũƄ ŘŪƎŠŌ ŉŔŧŌ Ɠž ūƏƈƆƈ ƇŧƂś Ŝŧţ ŧƂƅ

.ŕƎŗ řƂƆŸśƈƅŔ řƔŠƏƅƏƔŗƅŔ ŚŕƊƔŸƅŔƏ ŕƎśŕſƔƅƏśƏ řƔƏŧƗŔ ¿ƔƆţś Ɠž ĻƛŕŸž ĻŔũƏŧ

ř» ƆśƄƅŔ Žŕ» ƔųƈƏ � (UV) řƔŠŬ» ſƊŗƅŔ ƀƏž řŸŮƗŔ ƇŔŧŦśŬŕŗ ŘŪƎŠƗŔ ƋŨƍ ¿ŝƈƅ řƈŧŦśŬƈƅŔ řſƆśŦƈƅŔ ŽŮƄƅŔ ƀũųƉŏ

řŠƅŕŸƈƅŔƏ (ECD) ƓœŕƔƈƔƄƏũƎƄƅŔ ŽŮƄƅŔ Ƒƅŏ řžŕŲƙŕŗ (CL) řƔŬƊƈƏƔƆƅŔ ŜŕŸŗƊƛŔ ŉŕƔƈƔƄƏ Ə (FL)ūƊŬũƏƆſƅŔƏ (MS).¿ƔƆţśƅŔ ŖŗŬ ƏŌ ŽŧƎƅŔ ƑƆŷ ćŉŕƊŗ ŕƍũŕƔśŦŔ ƇśƔ řƊƔŸƆƅ ŽŰƏƅŔ řƂƔũųƏ řƂŗŬƈƅŔ

¿» ŝƈ ƐũŦƗŔ ŽŮƄƅŔ ƀũų ŕƈƊƔŗ řƔŠŬſƊŗƅŔ� ƀƏž řŸŮƗŔ ƇŔŧŦśŬŕŗ ŽŮƄƅŔ ƑƆŷ ŕƎśŕſƔƅƏś Ɠž řƔƏŧƗŔ ¿ƔƆţś Ɠž ŧƈśŷŔ

ƋŨ» ƍ Ŷ» Ƃś ŜƔţ řƔŠƏƅƏƔŗƅŔ ŚŕƊƔŸƅŔ Ɠž řƔƏŧƗŔ ¿ƔƆţśƅ ƇŧŦśŬś řƔƅŕŸƅŔ ŕƎśƔŬŕŬţƏ (ECD) Ə (CL) Ə (MS)Ə (FL).ƇŔũŻƏƄƔŗƅŔƏ ƇŔũŻƏũƄƔŕƈƅŔ ƉƔŗ ŕƈ ¿śƄƅŔ Ɛŧƈ ƉƈŲ řƔŬŕŬţƅŔ

�řƔŠƏƅƏƔŗƅŔ ŚŕƊƔŸƅŔƏ řƔœŔƏŧƅŔ ŕƎśŕſƔƅƏś Ɠž řƔƏŧƗŔ ŧƔŧţśƅ řƈŧŦśŬƈƅŔ (HPLC) ƀũųƅŔ řŮƁŕƊƈ řŸŠŔũƈƅŔ ƋŨƍ Ɠž Ƈś

Ɖƈ ŉŔƏŧƅŔ ŚƜŦŔŧśƏ řƄũţƏ řŬŔũŧƏ ¿ƔƆţś ¿ŕŠƈ Ɠž ŘŪƎŠƗŔ ƋŨƎƅ řƈŧƂśƈƅŔ ŚŕƂƔŗųśƆƅ űũŸśƅŔ Ƈś ƃƅŨ ƑƆŷ ŘƏƜŷƏ

.ƌƈƔƔƂś ¿ŠŔ

.ŚŕſƔƅƏśƏ řƔŠƏƅƏƔŗ ŚŕƊƔŷ řƔƏŧŌ� ŉŔŧƗŔ� řƔƅŕŷ ¿œŔƏŬƅŔ ŕƔžŔũŻƏśŕƈƏũƄ :ŗº ƃŒťƃŒ Řœº ƆƄƂƃŒ

____________________________________________

.2007/9/12 ƌƅƏŗƁ ťƔũŕśƏ 2007/9/5 ŜţŗƅŔ ƇƜśŬŔ ťƔũŕś

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hplc general

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