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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
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LIST OF TABLES
Table 1. comparisons parameters
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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
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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.
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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
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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 ).
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