seminar paper, 2006 pdf

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TABLE OF CONTENTS

TABLE OF CONTENTS ii

LIST OF TABLES iii

LIST OF FIGURES iv

1.INTRODUCTION 1

2. LITERATURE REVIEW 4

2.1 Structure of chloramphenicol (CAP) 4

2.2 Methods for Determination of CAP 5

2.3 Chromatographic analytical techniques of analysis 5

2.4 SPE-LC MS/MS Spectroscopy 5

2.5 Magnetic Sector and Quadrupole Mass analyzers 6

2.5.1 Magnetic Sector Msass Analyzer 7

2.5.2 Quadrupole Mass Spectrometer 73. METHODOLOGY 10

3.1 SPE-LC MS/MS Sample Preparation, Extraction and Principles 10

3.2 MSPD LC-MS/MS Sample Preparation, Extraction and Principles 10

3.3 SPE CAP extraction procedure from honey 13

3.4 MSPD CAP extraction from turtle tissue 14

3.5 MSPD Instrumental analysis 14

3.6 Analysis Results 16

3.7 SPE-LC-MS/MS Instrumental analysis 17

3.8 Analysis Result of SPE-LC-MS/MS 18

3.9 Quantification of CAP 18

3.10. General comparison of the instruments 19

4. DISCUSSION 20

4.1 Limit of detection (LOD) 20

4.2 Limit of Quantification (LOQ) 20

4.3 Accuracy 20

4.4 Repeatability: 20

4.5 Run time, cost and injection volume 21

4.6 Inclusivity 21

4.7 Raggedness and robustness 21

4.8 Scope of application of the instruments 21

5. SUMMARY 22

6.REFERENCES 24



iii

LIST OF TABLES

Table 1. comparisons parameters



iv

LIST OF FIGURES

Fig. 1 (A) CAP- tabulate and (B) CAP- capsule

Fig. 2 (A) Molecular structure of CAP and (B) the eight isomers of CAP

Fig.3. Schematic diagram of a magnetic sector mass analyser

Fig.4 (A) . Illustration of a quadrupole mass analyzer

Fig.4 (B). Quadrupole mass analyzer

Fig.5. Schematic representation of MSPD procedure

Fig. 6 . The set up of on-line MSPD-HPLC–MS/MS system

Fig.7 (A). CAP working standard solution

Fig. 7 (B). a blank soft-shelled turtle tissue sample spiked.

Fig. 8. MRM chromatograms of a blank honey sample with internal standard and the sample

spiked with internal standard CAP



1

1.INTRODUCTION

Safety of food and feed is one of the main objectives in consumer health policy. Maintaining a

high level of protection in this area is vital not only for public health but also to preserve

consumer confidence in food (regulation of the European Parliament and of the Council (EC) No

178/2002). One of the most important roles of modern analytical chemistry is probably the

assessment of food safety and food quality, especially those aspects related with the monitoring

of pesticides among other important residues or contaminants. Current concern about their

widespread and extensive use as well as their negative effects on human health has become the

main cause (Asensio et al., 2012).

Contamination of environment by xenobiotics is linked to industrialization and intensive

agriculture. Honey bees can be used for environmental monitoring as they are good biological

indicators, because of their mortality and residues present in their body or hive products (Zaneta

et al.,2011). Bees are subject to a number of diseases that affect their brood, with two of the most

serious being the larval bacterial diseases, called American and European foulbrood (AFB) and

Parasitic mite (varora jacobsoni) (Robert et al., 2008).

Antibiotics are widely used in animals for the treatment of diseases and also as animal growth

promoters. The use of antibiotics may lead to drug residues present in animal-derived foods; the

side effects of which would threaten public health (Wisanu et al., 2010). In most countries, few

antibiotics are allowed for use in combating these infections, with tylosin, oxytetracycline,

Sulfonamides and chloramphenicol CAP. Sulfonamides are effective against foulbrood, although

they are not permitted in many countries because of its implication that it has developed thyroid

tumors in mice and rats (Robert et al., 2008).

Chloramphenicol (CAP), which was first isolated from Streptomyces venezuelae in 1947, is a

broad-spectrum antibiotic that is widely used in animals for the treatment of several kinds of

infectious diseases because of its excellent antibacterial and well toleration (Tsuyoshi et al.,

2012; Wisanu et al., 2010; Li Yan et al., 2012; Voral et al., 2013). It is often used as a

prophylactic or disinfectant to prevent diseases or as a chemotherapeutic agent to control

diseases. It possessed broad spectrum antibacterial activity and used for the treatment of



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rickettsial and chlamydial diseases, gram +ve and gram –ve bacterial infections and topically for

superficial conjunctivial infections (Tyagi et al., 2012 ).

A number of corticosteroid and antibiotic combinations are frequently used as antibacterial

agents to cure infections particularly associated with the eye. These combinations are available in

different formulations including eye ointment, eye drops and ophthalmic suspensions.

Ophthalmic preparations of prednisolone acetate along with Chloramphenicol are widely

practiced for the treatment of superficial eye infections. However, this combination is not official

with British Pharmacopoeia or US Pharmacopoeia. But this medication is still employed in our

country Ethiopia.

However, the administration of CAP to humans, has induced various cytotoxic and genotoxic

effects such as chromosomal aberration and sister chromatid exchange in human lymphocyte

cultures, agranulocytosis, grey syndrome, aplastic anemia (bone marrow suppression) which is

not considered to be dose dependent, peripheral blood lymphocytes, an immortalized

lymphoblastoid cell line originating from human bone marrow exchange in lymphocyte cultures

(Alizadeh et al., 2012; Liu et al., 2010; Ligang et al., 2013; Tyagi et al., 2012).

Thus, since there is no safe limit or tolerance limit of CAP in food, any detectable amount of the

drug is reportable. Because of this, the use of CAP in food-producing animals, particularly in

aquaculture and honeybee has been prohibited in Europe, USA, India and China and it has been

also placed in Annex IV of European Council Regulation No. 2377/90 and strictly banned (Chen

et al., 2008; Tsuyoshi et al., 2011; Ligang et al., 2013). However, the European Commission has

defined a minimum required performance limit (MRPL) for CAP in food of animal origin at a

level of 0.3l gkg-1 (Commission Decision 2003/181/EC). Nevertheless, due to its low price and

consistent antibiotic effectiveness, illegal use of CAP still exists.

In 2001 and 2002, CAP residues were detected in various foodstuffs imported into the EU from

Asian countries (Tsuyoshi et al., 2012). It is still found in several animal-derived foods like

honey, due to its easy access, low cost, the great effect on the control of the bee infection, and

the increase in honey production. This had a major impact on international trade, and restrictions

were placed on the importation of these products. To monitor and control the compliance of a

zero tolerance level of CAP, sensitive, accurate and robust analytical methods are needed.



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Therefore, it is necessary to develop a sensitive and rapid method to control and monitor CAP

residues in food, such as honey and other aquatic animals is the question of analysts (Wisanu et

al., 2010; Li Yan et al., 2012).

Fig. 1 (A) CAP- tablate and (B) CAP- capsule

Up to date, our country Ethiopia used Chloramphenicol to combat a wide range of microbial

bacterial and infections including typhoid fever, meningitis, and certain infections of the central

nervous systems. In addition, it is also known that people are prescribed with chloramphenicol

eye ointments by physicians to treat superficial ocular infections involving the conjunctiva or

cornea, in topical ointments and to treat the external ear or skin, in various tablets for oral

administration, and in intravenous (i.v.) suspensions to treat internal infections yet. Thus, this

paper is to summarize and show how much CAP is a challenging antibiotic as well dangerous

medical drug used and by this to compare the most applicable analytical instruments (SPE-

LC/MS/MS and MSPD LC-MS/MS) used to determine its concentration in food and other

matrices.



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2. LITERATURE REVIEW

2.1 Structure of chloramphenicol (CAP)

CAP, with a chemical formula (C11H12Cl2 N2O5) was synthesized from the bacterium

Streptomyces venezuelae by David Gotlieb in 1947. This antibiotic is active against a wide

range of aerobic and anaerobic bacteria and fungi (FAO (2005). It has eight isomeric forms and

(A) Molecular structure of CAP.

Fig. 2 (A) Molecular structure of CAP and (B) the eight isomers of CAP

[(a) RR-p-CAP, (b) SS-p- CAP, (c) RS-p-CAP, (d) SR-p-CAP, (e) RR-m-CAP, (g) RS-m-CAP

and (h) SR-m-CAP]

as shown in Fig. 2 (B). Unless explained, the name generally refers to RR-p-CAP (levomycetin).

The SS-p-CAP (dextromycetin, DEX) and recemic mixture of the two is called synthomycin.

Among these isomers, only RR-p-CAP and SS-p-CAP exhibit antimicrobial property.

Comparing the two, the former is highly effective (Bjorn et al., 2011).



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2.2 Methods for Determination of CAP

Various applications of determination techniques have been reported for the analysis of CAP in

different samples matrices such as honey, milk, plasma, urine, seafood, shrimp, meat, egg,

feedwater, etc. by LC-MS/MS (Pavle et al., 2013; Li Yan et aL., 2012; Hao et al., 2011;

Rezende et al., 2011; Gaugain et al., 2009; Robert et al.,2008; Chicoa et al., 2008; Cronly et al.,

2010; Helene et al., 2006), MSPD-LC-MS/MS (Yanbin et al., 2012; Tsuyoshi et al., 2011),

LC -ESI- MS/MS (Vora1 and Raikwar. 2013; Rayane et al. 2007; Tyagi et al., 2008; Yves et al.,

2008 ), MIP-SPE-LC/MS/MS ( Martina et al. 2009; Brian et al., 2007; Martina and Libor ,

2009), MIP-CL (Wisanu et al., 2010) and magnetic molecularly imprinted polymer (MMIP)

(Ligang and Bin, 2013; Sara and Antonio, 2007; Hongyuan et al., 2013 ), Enzyme - Linked

Immuno sorbent Assay (BA-ELISA) (Wang et al., 2010), liquid chromatography–high resolution

mass spectrometry (LC–HRMS) (Hong et al., 2011), Solid phase micro extraction-Liquid

chromatography (SPME-LC) (Aresta et al., 2010) were among the various techniques used by

researchers. This shows that the determination of CAP is typical example of the most

challenging drug analysis which still needs fast, accurate and sophisticated analytical techniques.

2.3 Chromatographic analytical techniques of analysis

In spite of substantial technological advances in analytical field, most instruments cannot

directly handle such a complex sample matrixes yet. As a result, a sample-preparation step iscommonly involved before instrumental analysis. The main aim of sample preparation is to clean

up and concentrate the analytes of interest, while rendering them in a form that is compatible

with the analytical system (Mohammad et al., 2010).

2.4 SPE-LC MS/MS Spectroscopy

In the past few years many innovations in the analytical process that can be applied to extract

drugs, pollutants, and naturally occurring substances from food, environmental samples, and a

variety of biological have reported by most authors in this review. Many modern

chromatographic and electrophoretic instrumental techniques are sufficiently mature to enable

the hyphenation of different separation techniques with each other and with detectors that

provide a high information density. However, in many application areas, sample preparation is

still the bottleneck of such modern procedures. For example, in the analysis of complex semi-



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solid and solid matrices, particularly when the goal is to determine their trace components. The

complexity of most (semi-) solid environmental, food, and biological matrices makes exhaustive

sample treatment prior to proper separation-plus-detection mandatory (Ramos et al.,2012). In the

case of aqueous matrices and (some) biological fluids, the recent development allowed

completely on-line and automated sample treatment using solid-phase extraction- (SPE), solid-

phase micro-extraction- (SPME), or dialysis-based dedicated instrumentations.

Liquid chromatography coupled to a tandem quadrupole mass spectrometer (LC/MS-MS)

techniques are now widely used for screening purposes and these methods can cover a large

number of veterinary drugs (Gaugain et al., 2009; Hammel et al., 2008; Turnipseed et al., 2008).

The most common techniques in modern multi residue target pesticide analysis are gas

chromatography and liquid chromatography coupled to mass spectrometry (GC-MS, LC-MS)

and/or tandem mass spectrometry (GC-MS/MS, LC-MS/MS) with triple quadrupole mass

analyzers (Kmella et al., 2010).

Triple quadrupole MS/MS instruments are mainly applicable for sensitive and selective

quantitative measurements and the identification of known, targeted analytes in selected or

multiple-reaction monitoring (SRM or MRM) mode. A rapid, simple and sensitive multi-residue

method was developed and validated for the simultaneous quantification and confirmation of 69

pesticides in fruit and vegetables using liquid chromatography-tandem mass spectrometry (LC-

MS/MS) (Camino et al., 2010).

2.5 Magnetic Sector and Quadrupole Mass analyzers

Tandem mass spectrometry, abbreviated MS/MS, is any general method involving at least two

stages of mass analysis, either in conjunction with a dissociation process or in a chemical

reaction that causes a change in the mass or charge of anion. The most common tandem mass

spectrometric analyzer is used to isolate a precursor ion, which then undergoes spontaneously or

by some activation a fragmentation to yield product ions and neutral fragments.

A second spectrometer analyses is the product ions. The principle is illustrated in Fig.3 below.

The product ions spectrum will not display isotope peaks if the selected precursorm/z contains

only one isotope for each atomic species, which most often will be the case.



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2.5.1 Magnetic Sector Msass Analyzer

As the name suggests, these analyzers make use of a permanent magnet (or an electromagnet) to

separate the fragment ions. It consists of an evacuated curved metallic tube through which the

fragment ions pass on their way from the ion source to the detector. The electromagnets aremounted perpendicular to the tube and provide a stable and uniform magnetic field. As the ions

entering the analyzer have approximately the same kinetic energy. They have different velocities

depending on their masses; heavier ions would be slower. The field of the magnet makes these

ions to travel in a circular path generally of 60, 90 or 180 degrees. the ions with a different mass

to charge ratio will be detected at the detector. The mass to charge ratio of the fragment ions is

related to the parameters of the instrument as per the equation.

FIG.3 Schematic diagram of a magnetic sector mass analyser

2.5.2 Quadrupole Mass Spectrometer

A diagram of the quadrupole mass spectrometer is shown in Fig. 4 (A) and (B) below. Here, four

short, parallel metal rods (poles) with a diameter of about half a centimeter each are utilized.



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These rods are aligned parallel to and surrounding the fragment path as shown. Two nonadjacent

rods, such as those in the vertical plane, are connected to the positive poles of variable DC and

AC power sources, while the other two are connected to the negative poles. Thus, a variable

electric field is created, and as the fragments enter the field and begin to pass down the center

area, they deflect from their path. Varying the field creates the ability to focus the fragments one

at a time onto the detector slit, as in the magnetic sector instruments depicted. The quadrupole

instrument is newer and more popular since it is much more compact and provides a faster

scanning capability.

Fig.4 (A) Ilustration of a quadrupole mass analyzer.

Look at the following Fig. 4 (B) for simplicity to identify the path of the molecular ions.



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Fig..4 (B) Quadrupole mass analyzer



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3. METHODOLOGY

3.1 SPE-LC MS/MS Sample Preparation, Extraction and Principles

Méthodes for the extraction of pesticide residues in water and liquid matrices (milk, urine, blood,

serum, etc.) exploit the partitioning of the analytes between the aqueous phase and a nonpolar,

immiscible solvent or sorbent materials. However, liquid–liquid partitioning (LLP) has fallen

into disuse and has been replaced by SPE (Pico, 2012).

SPE extraction method is based on selective retention of the target analytes in a solid sorbent that

can then be eluted with an organic solvent. Several SPE materials have been developed from the

conventional alkyl-modified silica materials (C-18 non-polar phase) to the new materials based

on polymer sorbents that improve the retention of polar compounds . Although this technique

uses much less solvent than LLE, the volume can still be considered significant. Moreover, an

extra step of concentrating the extract to a small volume is needed. The demand to reduce

solvent volumes and to avoid using toxic organic solvents in LLE and SPE has led to substantial

efforts to adapt existing sample-preparation methods to the development of new approaches

(Cristina et al., 2011).

Since the mid-1970s, SPE has been one of the most popular techniques in sample preparation. It

is usually performed in a column/cartridge in order to remove interfering species. It comes in the

form of a packed syringe-shaped cartridge, a 96 well plate, or a 47 or 90 mm flat disk, each of

which can be mounted on a specific type of extraction manifold. The manifold allows multiple

samples to be processed by holding several SPE media in place and allowing for an equal

number of samples to pass through them simultaneously. A typical cartridge SPE manifold can

accommodate up to 24 cartridges, while a typical disk SPE manifold can accommodate six disks.

Most SPE manifolds are equipped with a vacuum port. Application of a vacuum speeds up the

extraction process by pulling the liquid sample through the stationary phase. The analytes are

collected in sample tubes inside or below the manifold after they passed through the stationary

phase. (Pico, 2012).

3.2 MSPD LC-MS/MS Sample Preparation, Extraction and Principles

Matrix solid-phase dispersion (MSPD) is a patent-protected process that was first introduced by

Barker in 1989 for the simultaneous disruption and extraction of semi-solid and solid samples.



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Several advantages associated with the use of SPE were its ability to avoid emulsions typically

formed in liquid–liquid extraction (LLE) or counter-current extraction, and the significant

reduction of the volume of solvent(s) required. This made Barker and his coworkers consider the

possibility of developing a similar approach for the preparation of solid samples. Direct

application of the homogenized tissues to the top of the SPE cartridge invariably resulted in the

column collapse due to plugging of the frit or upper column layers. Blending of the homogenized

tissues with diatomaceous earth yielded a semi-dried packing material that could easily be

packed in a column to be eluted as an SPE sorbent (Ramos et al., 2012).

As compared those enhanced extraction techniques, in which the extraction is carried out at high

pressures and/or temperatures or assisted by the application of a supplementary energy. In the

basic matrix solid-phase dispersion (MSPD) approach, the extraction process takes place under

ambient conditions and does not require any type of special equipment (Ramos et al., 2012).

MSPD is an SPE-based strategy in which a fine dispersion of the matrix is mixed with a sorbent

material (C-18, alumina, silica, etc.) with a mortar and a pestle. Usually, solid samples are

prepared for subsequent extraction and/or cleanup by a stepwise process that begins with the

disruption of the sample. After blending, the sorbent material is often packed into a mini column,

where the analytes are eluted by a relatively small volume of a suitable eluting solvent. Blending

is typically carried out with a glass pestle in a glass or agate mortar. New sorbents, such as the

coordination polymer [Zn(BDC)(H2O)2]n, have been developed, characterized, and tested for

MSPD. The new solid phase could be used in screening protocols by official regulatory

laboratories to identify pesticides in H. pectinata and other medicinal herbs (Pico, 2012).



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Fig.5 Schematic représentation of MSPD procedure

3.2.2 Application of MSPD technique

Matrix solid-phase dispersion extraction was applied to the extraction of sulfadiazine,

sulfamerazine, and sulfamethazine from human and animal bloods and CAP form honey. MSPD

has been developed for the determination of 16 OCPs in sludge from municipal sewage plants,

common pesticides and breakdown products (mostly pyrethroids and organochlorines) in cattle

feed polar pesticides in fruit juices, organophosphorus pesticides in bovine tissues, and pesticide

from onion. (Pico, 2012). MSPD can be performed using bonded silica (C-8 or C-18) or polar

materials (e.g., Florisil, silica, alumina) multiclass analysis of pesticides in the medicinal herb

Hyptis pectinata. Results showed that [Zn(BDC)(H2O)2]n can be successfully used for analysis

of pyrimethanil, ametryn, dichlofluanid, tetraconazole, flumetralin, kresoxim-methyl, and

tebuconazole in medicinal herbs.. The separation and determination of the analytes were carried

out by high-performance liquid chromatography(Yupu et al., 2012).



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3.3 SPE CAP extraction procedure from honey

Honey spiked with internal standard D5-CAP was heated to 50ºC and

dissolved in deionized water and reheated for complete dissolution.

To this mixture ACN and NaCi was added, then agitated , and finally

centrifuged.

The top organic layers were then transferred to polypropylene tubes

(15 ml) and evaporated (50ºC) to 6ml under nitrogen.

Hexane was added and this was vortexed, the layer was discarded and

the extracts were evaporated to dryness at 50ºC under a nitrogen stream.

The solid obtained was then reconstituted in (water :ACN) ratio (95

: 5 ) of 200 ml and filtered through 0.2 mm PVDF syringe filters.

An aliquot (10 ml) was injected onto the HPLC column HPLC

column.



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3.4 MSPD CAP extraction from turtle tissue

Soft-shelled turtle tissue was homogenized using an electric blender.

The homogenized tissue , D5-CAP working solution and C18 were gently

blended with agate glass mortar until a homogeneous mixture

was obtained.

After being dried at room temperature, the MSPD blend was laboratory-

packed into the extraction vessel fitted to HPLC-MS/MS

On-line MSPD-LC–MS/MS was carried out

3.5 MSPD Instrumental analysis

MSPD-ultra-fast LC–MS/MS system was, a P230 high pressure pump (Elite, Dalian, China)

equipped with a 6-port switching valve was used to re-circulate the extraction solvent for

extracting constantly. The customized MSPD process was performed by a 25 x 10 mm i.d.

extraction vessel (Michrom Bioresources, Auburn, CA) and on-line coupled with LC/MS/MS bya 10-port switching valve (VICI, Schenkon, Switzerland)

Chromatographic analysis was performed on a Waters 2695 LC system (Waters, Milford, MA,

USA) which was equipped with a quaternary pump, an autosampler, a vacuum degasser and a

LC workstation. The analytes separation was achieved on a Halo core-shell C-18 silica column

(50 x2.1 mm, 2.7 lm; Advanced Materials Technology, USA).

A triple-quadrupole linear ion trap mass spectrometer (4000Q-Trap, Applied Biosystems, Foster

City, CA) equipped with an electro-spray ionization (ESI) was used in negative ionization

multiple-reaction monitoring (MRM) mode. The prepared and MSPD paced sample has carefully

fitted to the HPLC-MS/MS system a gradient elution system was applied for separation

employing mobile phase A (0.1% aqueous formic acid solution) and mobile phase B (ACN:

water) in the ratio of (80:20 v/v). the gradient profile had been carried out starting from 10-50%



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B in 2 min, then to 100% B in 5 min held for 2 min and the to 10% B in 0.5 min at a flow rate of

0.4 mL min-1 at a column temperature set to 30ºC and injection volume of 5µL. (Yanbin Lu et

al., 2012)

Fig. 6 The set up of on-line MSPD-HPLC–MS/MS system

Limit of detection (LOD) and limit of quantitation (LOQ)

The LOD and LOQ were considered as the analytes' minimum concentrations that can be

confidently identified and quantified by the method, respectively. The LOD was determined by

analysing blank sample at levels that provided signals at three times above the background

noises. In a similar way, the limit of quantization (LOQ) was identified at signal to noise ratios

equaled to ten. The calculated critical concentrations LOD and LOQ for the screening. Precisionof the method was evaluated by measuring intra-and inter-day relative standard deviations

(RSDs). The intra-day precision was evaluated by repeated analyses of CAP at five different

fortified concentrations on three sequential runs in six replicates. The intra-day precision was

performed by analyzing spiked samples over 6 days (Yanbin et al., 2011)



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3.6 Analysis Results

Fig.7 (A) CAP working standard solution

Fig. 7 (B) a blank soft-shelled turtle tissue sample spiked.

Quantification of unknown is give by : Ci =

As = concentration of the standard

Ai x CsAs

where: Ai = area of unknown sample peak

Cs = total area of the standard peak



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3.7 SPE-LC-MS/MS Instrumental analysis

The LC–MS/MS system consisted of a Series 200 pump and autosampler (Perkin Elmer,

Norwalk, CT, USA) coupled to an API 2000 triple quadrupole mass spectrometer (Sciex,

Toronto, Canada). The chromatographic separation was performed on a LUNA ODS(2) C18

3_m column, 7.5mm×4.6mm i.d. ( Phenomenex, Torrance, CA, USA), by using an isocratic

mobile phase of methanol-5mM ammonium acetate (60:40, v/v). The flow rate was set at 0.2 ml

min−1, the injection volume at 50µL and the column temperature at 40◦C. The octadecyl (C18)

solid-phase extraction (SPE) cartridges (500 mg/3 ml) were from J.T. Baker (Yanbin et al.,

2012).

Two calibration curves were constructed by plotting the area ratio of m/ z 321 → 152 versus 326

→ 157 and m/ z 321 → 257 versus 326 → 262 against their corresponding amount ratiotransitions monitored and were compared by a Student’s t-test to find any significant difference

between the series.. The ion 157 from D5-CAP were used as internal standard for both CAP ions

152 and 194, while 262 was used as internal standard for 257. The EU-decision 657/2002/EC

also suggests another approach in calculating CCα and CCβ. This is the so called calibration

curve procedure where the concentration corresponding to the y-intercept plus 2.33 times the

standard error at the intercept equals the decision limit. The CCβ is then calculated as the

concentration at the decision limit plus 1.64 times the standard deviation at the decision limit

(Helene et at., 2006; Tyagi et al., 2008).



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3.8 Analysis Result of SPE-LC-MS/MS

Fig. 8 MRM chromatograms of a blank honey sample with internal standard and the sample

spiked with internal standard CAP.

3.9 Quantification of CAP

CAP quantitation was accomplished by the isotope dilution method considering the most intense

ion transition (Helene et al., 2006).



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Quantification of unknown is give by : Ci = Ai x Cs

As

where: Ai = area of unknown sample peak

Cs = total area of the standard peak

As = concentration of the standard

LOD is given by, CCα = 2 .33σ N and LOQ = CCα + 1 .64σ S

3.10. General comparison of the instruments

,

a a

Table 1. comparisons parameters

Comparison parameters SPE-LC-MS/MS MSPD LC-MS/MS referencesSensitivityLODLOQ

0.070 0 (µg Kg-1)0.100(µg Kg-1)

0.0750 ng0.250 ng

Yanbin et al., 2012Brian et al.,2007

Accuracy (R2) 0.9996 0.9993 Brian et al.,2007Yanbin et al., 2012

Repeatability 100.204.60

:Recovery(R %)Precision (RSD)

98.072.73

Brian et al.,2007Yanbin et al., 2012

Total run time (min) > 1 hr 15 » »

Cost High Relatively low cost L Ramos, 2012,Lina et al., 2009Yanbin et al., 2012,Yupu et al., 2012

Flow rate (mL min-1 or

µL min-1

0.2 mL mi-1 10 µL min-1 » »

50.0 µLSample economy:Injection volume (µL) 5.0 µL » »

Inclusivity: Inclusive

applicability of theMethods to detect the analytesat standard level

Inclusive » »

Raggedness Ragged Ragged » »

Robustness Robust Robust » »Scope of application Food, drugs, human blood, urine andserum

All solids and semi-solidenvironmental and biological samples, pollutants, drugs, andeven bacteria components

L Ramos, 2012,Lina et al., 2009Yanbin et al., 2012,Yupu et al., 2012Yupu. B et al., 2007



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4. DISCUSSION

CAP, a broad spectrum anti biotic drug contaminated foods of animal and plant origin by

different ways has been listed in an annex 1 if the decision council 96/23/EC31 for which a zero

tolerance residue limit established. Although many types of analytical methods were applied to

investigate its level in different sample matrices, SPE-LC-MS/MS and MSPD-LC-MS/MS

chromatographic techniques were selected in this review to be compared.

4.1 Limit of detection (LOD)

The limit of detection for SPE-LC-MS/MS and MSPD-LC-MS/MS was reported 0.0070 µg Kg-1

and 0.0075 ng Kg-1 respectively. It verifies that the values obtained by signal-to-noise and blank

détermination méthodes MSPD showed higher detection capacity than SPE instrument

4.2 Limit of Quantification (LOQ)

The amount of CAP quantitatively determined with suitable precision and accuracy (LOQ) for

SPE instrument was given 0.100 µg Kg-1 and that for MSPD was 0.250 ng Kg-1. this result

showed, MSPD-LC-MS/MS instrument has better quantification capacity than SPE LC-MS/MS

4.3 Accuracy

Linearity evaluation regression (R 2) value for SPE-LC-MS/MS was 0.9996 which showed that

the instrument is accurate to be applied in CAP determination. similarly the linearity test for

MSPD-LC-MS/MS was 0.9993 which attributed that it was also at good accuracy and efficient

method. The Recovery (R %) test comparison showed 100.20 for the SPE -LC-MS/MS

method and 98.07 for the MSPD -LC-MS/MS technique. This also tells that the instruments'

respose " y" linearly related to the standard concentration " x" for a limited range of

concentrations so that both showed good accuracy.

4.4 Repeatability:

The (RSD) values were 4.60 and 2.73 respectively. This verify that the instruments MSPD -LC-

MS/MS has greatest performance than SPE-LC-MS/MS. In contrast, as it has been shown above,

SPE-LC-MS/MS has greater performance in quantifying CAP from matrices.



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4.5 Run time, cost and injection volume

Time, total analysis cost and sample economy are required parameters to compete analytical

instruments. Based on this fact, SPE-LC-MS/MS took more than an hour, used 50 µL injection

volume and is relatively subjected high analysis cost. On the other hand, in MSPD -LC-MS/MS

analysis can be completed only within 15 min. Besides, it has used only 5 µL injection volume

which make it very sample economical and has relatively low analysis coast than the former.

4.6 Inclusivity

Both the applied methods were so inclusive that they are applicable for determination of CAP at

the aim of Commission Decision 2003/181/EC requirements (Yanbin et al., 2012; Brian et al.,

2007).

4.7 Raggedness and robustness

By performing the analysis of aliquots from homogeneous lots in different laboratories, . both

researchers (Brian et al., 2007 and Yanbin et al., 2012) explicitly have shown that the two

instruments are Ragged and robust i.e the methods were not influenced by a minor change in

experimental conditions.

4.8 Scope of application of the instruments

SPE-LC-MS/MS determination of CAP applied and validated for analysis of Food, drugs,

human blood, urine and serum (Brian et al., 2007 ; Yanbin et al., 2012 and Cronly et al.,2010).

consequently, MSPD -LC-MS/MS technique has been validated for wider range of samples such

as for all solids and semi-solid environmental and biological samples, pollutants, drugs, and

even bacteria components This greatly acertain that, the later method has the greater application

area.



22

5. SUMMARY

With growing concerns over food safety and the need to increase sample-throughput in analytical

testing laboratories, there is a constant requirement for accurate, simpler, faster and improved

analytical methods. The complexity of food matrices and the presence of much potential

interference, require specific and selective methods of analysis. Often LC–MS/MS is used to

provide the specificity needed in order to correctly identify contaminants in food samples.

However, LC–MS/MS alone does not provide the sensitivity and accuracy frequently required by

regulatory and food safety agencies. A common technique used to complement LC–MS/MS

analysis is the pretreatment of the sample by clean-up methods such as solid-phase extraction

(SPE),MIP-SPE preconcentration, liquid–liquid extraction (LLE), supercritical fluid extraction;

etc. clean-up techniques remove many of the matrix interferences allowing more sensitive and

accurate analysis by an LC–MS/MS methods. (Brian et al., (2007).

A quantitative SPE- LC-MS/MS method for determination of CAP in honey at trace levels has

been reported, entailing aqueous dissolution of the matrix to liberate the residues, followed by

SPE extraction and a final liquid–liquid partitioning step. The result obtained by this method

was validated at the aim of meeting Commission Decision 2002/657/EC requirements EU

(Brian et al., (2007).

To ensure the absence of chemical contamination in honey, screening methods using rapid testkits are regularly employed, and positive results were further confirmed by a confirmatory

technique. Recently, Rapid, effective and efficient technique for the analysis of trace substances,

both exogenous (drugs, pollutants, pesticides) and endogenous ones (food and bacteria

components, etc.) from solid, semi-solid, and viscous matrices (animal tissues), blood, milk,

bacteria, fruits, vegetables, etc , using on-line MSPD-LC–MS/MS has been introduced which is

highly sensitive, fast, accurate, more precise and more scope of application area than SPE-LC-

MS/MS ( Ramos, 2012 ; Lina et al., 2009 ; Yanbin et al., 2012, Yupu et al., 2012 and Yupu.

et al., 2007).

Moreover, MSPD-LC-MS-MS method is better in reducing the amount of chemical waste

generated due to the hazardous organic solvents used in the analysis of CAP and other

antibiotics than the other. It has also the highest ability to detect the targeted analytes from a

wide range of sources and has higher capability of isolating the amount of the sample interest in



23

nanoscale unit (ng kg-1 level) from environment, food and other biological matrices, (Yanbin et

al., 2012).

In addition, various sorbents like reversed phase materials, non-retentive supporting materials,

MIPs, emerging supporting materials, functional carbon nanotubes can be employed for sample

dispersion and analyte extraction. For these reasons, the on-line MSPD-LC-MS/MS method is

obviously a more economical and sustainable method for green analytical chemical analysis with

a bright future. (Yanbin et al., 2012 , Sara et al., 2007;Yupu et al., 2012 ; Ramos, 2012 ).



24

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