Quantification of meropenem in plasma and cerebrospinal fluid by micellar electrokinetic capillary chromatography and application in bacterial meningitis patients

A high-performance micellar electrokinetic capillary chromatography (MEKC) has been demonstrated for the determination of meropenem in human plasma and in cerebrospinal fluid (CSF) and application in meningitis patients after intravenous (IV) administration. Plasma sample was pretreated by means of solid-phase extraction (SPE) on C(18) cartridge and CSF sample was by direct injection without sample pretreatment, with subsequent quantitation by MEKC. The separation of meropenem was carried out in an untreated fused-silica capillary (40.2 cm x 50 microm I.D., effective length 30 cm) and was performed at 25 degrees C using a background electrolyte consisting of Tris buffer (40 mM, pH 8.0) solution with sodium dodecyl sulfate (SDS) as the running buffer and on-column detection at 300 nm. Several parameters affecting the separation and sensitivity of the drug were studied, including pH, the concentrations of Tris buffer and surfactant. Using cefotaxime as an internal standard (IS), the linear ranges of the method for the determination of meropenem in plasma and in CSF were all over 0.5-50 microg/mL; the detection limits (signal-to-noise ratio=3) of meropenem in plasma and in CSF were 0.2 microg/mL and 0.3 microg/mL, respectively.     

Quantitative determination of nitroglycerin in human plasma by capillary gas chromatography negative ion chemical ionization mass spectrometry

A quantitative method for determination of nitroglycerin in human plasma was developed. Nitroglycerin and the internal standard (butane-1,2,4-triyl trinitrate) were extracted from plasma with pentane. The extracts were analysed by gas chromatography mass spectrometry using fused silica capillary columns and electron capture negative ion chemical ionization. The quantitation limit of the method was about 50 pg ml-1. Linear calibration curves were obtained in the range of 50-1600 pg ml-1. Precision at the level of 100 pg ml-1 was 4%.     

Validation of a rapid micellar electrokinetic capillary chromatographic method for the simultaneous determination of isoniazid and pyridoxine hydrochloride in pharmaceutical formulation

An efficient and reliable micellar electrokinetic capillary chromatography (MEKC) method has been developed for the simultaneous determination of isoniazid (ISO) and pyridoxine hydrochloride (PYR) in pharmaceutical formulations. A chemometric two level full factorial design approach was used to search for the optimum conditions of separation. Three parameters were selected for this study: the buffer pH, the buffer concentration and sodium dodecyl sulphate (SDS) concentrations. Resolution, peak symmetry and analysis time were established as response. The two analytes were separated within 6 min with the optimized conditions: 50 mM borate buffer, 25 mM SDS pH 7.8, 35 degrees C, at 50 mbar 4s injection and 30 kV by using a fused silica capillary (72 cm effective length, 50 microm i.d.). The detection wavelength was set to 205 nm. Meloxicam was used as internal standard. The method was validated with respect to stability, linearity range, limit of quantitation and detection, precision, accuracy, specificity and robustness. The detection limits of the method were 1.0 microg mL(-1) for ISO and 0.40 microg mL(-1) for PYR and the method was linear at least in the range of 3.0-100 microg mL(-1) for ISO and 1.0-100 microg mL(-1) for PYR with excellent correlation coefficients (0.9995 for ISO and 0.9998 for PYR). Relative standard deviations (R.S.D.s) of the described method ranged between 0.54 and 2.27% for intra-day precision and between 0.65 and 2.69% for inter-day precision. The developed method was applied to the tablet form of ISO and PYR-containing the pharmaceutical preparations and the data were compared with obtained from the standard addition method. No statistically significant difference was found.     

Gas chromatography with nitrogen-selective detection of metoprolol from human plasma after reaction with phosgene

A method for the determination of therapeutic levels of metoprolol in human plasma is presented. Metoprolol and the internal standard are extracted from the buffered plasma sample to an organic phase containing 4 x 10^?3M phosgene. After 10 min the organic phase is taken to dryness. The residue is dissolved in ethyl acetate and the formed oxazolidine derivatives are analyzed by gas chromatography with nitrogen-selective detection. With packed columns, rectilinear standard curves through the origin were obtained down to 80 nmoles/l of plasma. The precision of the method at 200 nmoles/l was 1.5% (n = 8). The sensitivity of the method was improved by using capillary column gas chromatography. Linear standard curves were obtained down to 10 nmoles/1 of metoprolol in plasma. The precision of the method at the 50 nmoles/1 level was 2.2% (n = 7). With this simple and straightforward method using extractive derivatization 30 samples can be handled in a day.     

Simultaneous determination of paracetamol, caffeine and propyphenazone in ternary mixtures by micellar electrokinetic capillary chromatography

A new micellar electrokinetic capillary chromatographic method has been developed to analyze the pharmaceutical preparations containing ternary combination of paracetamol (PAR), caffeine (CAF) and propyphenazone (PRO). Best results were obtained by using 20mM pH 9.0 borate buffer containing 30mM sodiumdodecylsulphate as the background electrolyte. Diflunisal (DIF) was used as internal standard (IS). The separation was performed through a fused silica capillary (50microm internal diameter, 44cm total length, 35.5cm effective length) at 25 degrees C with the application of 3s of hydrodynamic injection at 50mbar pressure and a potential of 29kV. Detection wavelength was 200nm. Under these conditions, the migration times were found to be 5.174min for PAR, 5.513min for CAF, 7.195min for DIF, and 9.366min for PRO. Linearity ranges for the method were determined as 2-200microgmL(-1) for PAR and CAF and 3-200microgmL(-1) for PRO. Limit of detections were found as 0.6microgmL(-1) for PAR and CAF and 0.8microgmL(-1) for PRO. According to the validation study, the developed method was proved to be accurate, precise, sensitive, specific, rugged and robust. Three pharmaceutical preparations, which are produced by different drug companies in Turkey, were analyzed by the developed method. One of the same preparations was also analyzed by the derivative ratio spectro zero-crossing spectrophotometric method reported in literature. No significant differences were found statistically.     

Field-amplified sample stacking in capillary electrophoresis for the determination of clozapine, clozapine N-oxide, and desmethylclozapine in schizophrenics' plasma

A method of field-amplified sample stacking in capillary electrophoresis is described for the simultaneous determination of clozapine (CZP) and its metabolites, clozapine N-oxide (CNO), and desmethylclozapine (DMC), in human plasma. Plasma (0.2 mL) was extracted with organic solvents (ethyl acetate/n-hexane/isopropyl alcohol, 8/1/1 by volume) and centrifuged. An aliquot of supernatant was evaporated and suitably reconstituted with water for CE analysis. An untreated fused-silica capillary was used (31.2 cm; effective length, 20 cm; 50 microm i.d.) for the analysis. The background buffer was phosphate buffer (400 mM, pH 3.0) containing 50% ethylene glycol. The separation voltage was 25 kV with a detection wavelength of 214 nm. In the method validation, the calibration curves were linear (r > or = 0.98) over a range of 50-800 ng/mL for CZP, 30-180 ng/mL for CNO, and 25-600 ng/mL for DMC. The relative standard deviation (R.S.D.) and relative error (R.E.) were all less than 11% for the intra- and inter-day assays. The limits of detection (S/N = 3, electric-driven injection, 99.9s) of CZP, DMC, and CNO were 5, 5, and 10 ng/mL, respectively. After continuing treatment with the CZP tablets, a blood sample from one male schizophrenic patient (41-year-old, 62 kg) who had been receiving ongoing treatment with the CZP tablets was prepared and analyzed. The levels of CZP, DMC, and CNO were determined and the feasibility of the method's application in clinical treatment was proven.     

Separation of carbamazepine and five metabolites,and analysis in human plasma by micellar electrokinetic capillary chromatography

A rapid and feasible method was developed for the analysis of carbamazepine and its five metabolites (10,11-dihydro-10,11-epoxycarbamazepine, 10,11-dihydro-10,11-dihydroxycarbamazepine, 10,11-dihydro-10-hydroxycarbamazepine, 2-hydroxycarbamazepine and 3-hydroxycarbamazepine) in human plasma. Separation of the analytes is based on micellar electrokinetic chromatography, in untreated fused-silica capillary (48.5/40.0 cm length, 50 microm I.D.) with phosphate buffer (30 mM, pH 8.00) as background electrolyte, containing 50 mM sodium dodecylsulfate, and methanol (15%, v/v) as organic modifier. Clean up of human plasma samples was carried out by means of a solid-phase extraction procedure, which gave a high extraction yield for all six carbamazepines (>88%). The overall precision of the method gives a mean RSD of about 1.8%. The limit of quantitation for all analytes is < or = 0.30 microg ml(-1), the limit of detection < or = 0.12 microg ml(-1).     

Validation of methyl malondialdehyde as internal standard for malondialdehyde detection by capillary electrophoresis

The aim of this study was to validate, by capillary electrophoresis, the use of synthesized methyl malondialdehyde as the internal standard for the direct quantification of free and total (free+bound) malondialdehyde in biological samples. All analyses were performed in 20 cm x 50 microm uncoated capillaries at 20 degrees C, using 25 mmol/L borax (pH 9.3) and 5 mmol/L tetradecyltrimethylammonium bromide as running buffer. The applied voltage was -4kV (about 8 microA), the detector being set at 260 nm for a total run time of 8 min per sample. Free malondialdehyde was evaluated after acetonitrile extraction, while the samples evaluated for total malondialdehyde were, before extraction, hydrolyzed for 1h at 60 degrees C in the presence of 1 mol/L NaOH. The detection threshold was 0.2 micromol/L in microsomes and 0.4 micromol/L in plasma. As an application of the method, three pools of rat liver microsomes were quantified before (0.35+/-0.1 and 1.1+/-0.5 nmol/mg protein, free and total malondialdehyde, respectively, mean+/-SD) and after lipoperoxidation induction using systems able to generate oxygen free radicals (18.4+/-3.2 and 19.7+/-2.0 nmol/mg protein). The results were confirmed by isotopic dilution gas chromatography-mass spectrometry, used as the reference method. The feasibility of capillary electrophoresis for malondialdehyde determination in normal and pathological human plasma was also investigated.     

Solid-phase microextraction with capillary gas-liquid chromatography and nitrogen-phosphorus selective detection for the assay of antidepressant drugs in human plasma

Solid-phase microextraction (SPME) was tested as a sample preparation for the simultaneous assay of ten antidepressant drugs and metabolites (TADs) in human plasma. Aqueous NaOH (0.5 ml, 1 M) and chloramitriptyline (50 μl, 40 μg/ml) as internal standard (I.S.) were added to a 2-ml plasma sample. This mixture was extracted with a 100-μm polydimethylsiloxane SPME fiber (Supelco) for 10 min. After washing in water and methanol (50%) and subsequent drying at room temperature, desorption of the fiber was performed in the injection port of a gas chromatograph at 260°C for 1 min (HP 5890, DB-17 30 m×0.25 mm I.D., 0.25 μm capillary; 0.7 ml/min nitrogen; nitrogen-phosphorus selective detection). The recovery was found to be very low from plasma (0.3% to 0.8%) but considerably higher from water (about 15%). Therefore, the high protein binding of antidepressants appears to be the main limiting mechanism for a better extraction. However, the analytes were well separated and the calibrations were linear between 125 ng/ml and 2000 ng/ml. The limits of quantification were about 90 ng/ml for imipramine and desipramine, 125 ng/ml for amitriptyline, trimipramine, doxepine, nortriptyline and mianserine and about 200 ng/ml for maprotiline, clomipramine and desmethylclomipramine. The recovery was improved by increasing the extraction time. The influence of the concentrations of the sum of proteins and of α-acid glycoprotein on the peak-area ratios A_TAD/A_I.S. and on absolute peak areas was studied. Peak-area ratios increased with decreasing protein concentration but were found to be independent on α-acid glycoprotein. A simple model for the explanation of the effect is presented. Measures for the improvement of sensitivity are discussed. As presented in this paper, which first describes SPME for the analysis of drugs in plasma, SPME with a short extraction time can be of only very limited value for therapeutic drug monitoring. Lower concentrations than the limit of quantification are usually found at therapeutic doses. The method can be useful for toxicological analysis after the accidental or suicidal intake of higher doses. However, an about 10-fold improvement of the sensitivity of the method seems to be possible.     

Determination of amikacin in human plasma by high-performance capillary electrophoresis with fluorescence detection

A selective and reproducible high-performance capillary electrophoretic (HPCE) method for the quantification of amikacin (AMK), an aminocyclitol antibiotic, in human plasma, has been developed for use in clinical laboratory tests. The method involves ultrafiltration (UF) of plasma before derivatization with the fluorescence derivatization reagent 1-methoxy-carbonylindolizine-3,5-dicarbaldehyde at room temperature for 15 min in the dark. An aliquot of the derivatives is directly introduced into the fused-silica capillary [75 cm (effective length)×50 μm I.D.] at the anode side by dynamic compression injection (50 hPa for 6 s). After electrophoresis with 40 mM SDS-20 mM phosphate-borate buffer (pH 7) in the micellar electrokinetic chromatography (MEKC) mode at 30 kV, the derivative had a retention time of 16.7 min and was detected by fluorescence intensity at 482 nm (with irradiation at 414 nm). The precision (n = 5) of the method is 4.08 and 1.59% (C.V.) at the 50 and 100 μg AMK/ml plasma levels, respectively. Linearity (r = 0.998) was established over the concentration range 5–100 mg of AMK/ml plasma and the detection limit (at a signal-to-noise ratio of 3) is 0.5 μg AMK/ml plasma. This assay method could potentially have wider application in the determination of other aminocyclitol antibiotics, such as arbekacin, dibekacin, kanamycin, in human plasma as well as of AMK.     

Simultaneous determination of iodate and periodate by capillary zone electrophoresis: application to carbohydrate analysis

Iodate and periodate were rapidly (in 11 min) separated from each other with high column efficiency by capillary zone electrophoresis, using a fused silica tube (50 microns i.d., 80 cm) and 100 mM acetate buffer, pH 4.5, as carrier. On-column uv detection at 222 nm allowed sensitive detection down to the picomole level, and measurement of relative peak area to that of pyromellitic acid (internal standard) enabled reproducible determination of these ions. This method was proved useful for periodate oxidation analysis of various carbohydrates.     

Simple GC-FID method development and validation for determination of alpha-tocopherol (vitamin E) in human plasma

This paper describes the development and validation of a novel GC-FID method for the determination of alpha-tocopherol concentration in human plasma which does not requires derivatization. The standard solutions and the plasma working solutions were prepared in absolute ethanol. To determine the concentration of alpha-tocopherol in human plasma, an aliquot of the plasma sample was deproteinized with ethanol. alpha-tocopherol was extracted with a mixture of hexane and dichloromethane (9:1). GC separation was performed using a HP-5 capillary column. Nitrogen was used as carrier gas at a flow-rate of 2 ml min(-1). Calibration curves were linear over the concentration range 1-30 microg ml(-1) (for standard solutions and solutions without endogenous alpha-tocopherol in plasma) and 5-34 microg ml(-1) (for solutions with endogenous alpha-tocopherol in plasma). Absolute recovery, precision, sensitivity and accuracy assays were carried out. The analytical recovery of alpha-tocopherol from plasma averaged 97.44%. The limit of quantification (LOQ) and the limit of detection (LOD) of method for standard samples were 0.35 microg.ml(-1) and 0.30 microg.ml(-1), respectively. Within-day and between-day precision, expressed as the relative standard deviation (RSD) were less than 4%, and accuracy (relative error) was better than 8%. This novel method, developed and validated in our laboratory, could be successfully applied to the in-vivo determination of alpha-tocopherol. The endogenous alpha-tocopherol amounts in blood of twelve healthy volunteers with no vitamin drug usage were measured with this method.     

Detection of a mixed-backbone oligonucleotide (GEM 231) in liver and tumor tissues by capillary electrophoresis

A simple, rapid and sensitive method has been developed and validated for the analysis of a mixed-backbone oligonucleotide (GEM 231) in tumor tissues. The analysis was performed using a capillary electrophoresis (CE) system with UV detection. An extended light path (bubble cell) capillary column of 64.5 cm (effective length 56 cm)×50 μm I.D. is used as the separation column. The optimized chromatographic conditions were background electrolyte: sodium borate buffer (60 mM, pH 9.1), electrokinetic injection: 10 s, applied voltage: 30 kV, detection at λ=210 nm. A linear relationship was observed between the peak area and the amount of GEM 231 in the range of 1.0–1000 μg/ml. The lower detection limit of the drug was 100 pg with an average recovery of about 75±5%. The inter-day and intra-day relative standard deviations were <10%. Assay validation studies revealed that CE method is reproducible and specific for the determination of GEM 231 in tissue homogenates with a run time of less than 5 min.     

Development and validation of a capillary electrophoresis method for the characterization of herpes simplex virus type 1 (HSV-1) thymidine kinase substrates and inhibitors

A fast, convenient capillary electrophoresis (CE) method was developed for monitoring the enzymatic reaction of herpes simplex virus type 1 thymidine kinase (HSV-1 TK). The reaction was performed in a test tube followed by quantitative analysis of the products. The optimized CE conditions were as follows: polyacrylamide-coated capillary (20 cm effective length x 50 microm), electrokinetic injection for 30s, 50 mM phosphate buffer at pH 6.5, constant current of -60 microA, UV detection at 210 nm, UMP or cAMP were used as internal standards. Phosphorylated products eluted within less than 7 min. The limits of detection were 0.36 microM for dTMP and 0.86 microM for GMP. The method was used to study enzyme kinetics, and to investigate alternative substrates and inhibitors.     

Pre-analytical factors affecting ascorbic and uric acid quantification in human plasma

We have recently described a new capillary electrophoresis assay to measure serum ascorbic and uric acids in which a baseline separation of peaks was obtained in less than 4 min by using a 60.2 cm x 75 microm uncoated capillary with a 100 mmol/L sodium borate running buffer pH 8. Since during sample preparation AA is rapidly oxidized, we employed our new capillary electrophoresis method to analyze the pre-analytical factors affecting its stability. In particular we evaluated how the standard mix preparation, the blood collection (plasma EDTA or serum) and the plasma protein precipitation influence the results of analysis. Our data suggest that standard ascorbate must be dissolved in a solution containing cysteine and EDTA in order to avoid oxidation and that EDTA blood collection is better than serum for AA measurement. Moreover, the type and the quantity of the precipitating compound are critical parameters to obtain a complete recovery of analytes. We performed AA and UA analysis in 32 healthy volunteers with the optimized experimental conditions by using our capillary electrophoresis method and a reference CE assay. Obtained data were compared to Bland-Altman test to verify the accuracy of our CZE method.     

Combining poly (methacrylic acid-co-ethylene glycol dimethacrylate) monolith microextraction and on-line pre-concentration-capillary electrophoresis for analysis of ephedrine and pseudoephedrine in human plasma and urine

A method based on poly (methacrylic acid-co-ethylene glycol dimethacrylate) (MAA-EGDMA) monolith microextraction (PMME) and field-enhanced sample injection (FESI) pre-concentration technique was proposed for sensitive capillary electrophoresis-ultraviolet (CE-UV) analysis of ephedrine (E) and pseudoephedrine (PE) in human plasma and urine. The PMME device consisted of a regular plastic syringe (1 mL), a poly (MAA-EGDMA) monolithic capillary (2 cm x 530 microm I.D.) and a plastic pinhead connecting the former two components seamlessly. The extraction was achieved by driving the sample solution through the monolithic capillary tube using a syringe pump, for the desorption step, an aliquot of organic solvent, which normally provided an excellent medium to ensure direct compatibility for FESI in CE, was injected via the monolithic capillary and collected into a vial for subsequent analysis by CZE. The best separation was achieved using a buffer composed of 0.1M phosphate electrolyte (pH 2.5) and 10% acetonitrile (v/v). The combination of both pre-concentration procedures allowed the detection limits of the analytes down to 5.3 ng/mL and 8.0 ng/mL in human plasma and urine, respectively. Excellent method of reproducibility was found over a linear range 50-5000 ng/mL in plasma and urine sample. Plasma and urine samples from volunteers receiving pseudoephedrine have also been successfully analysed.     

Capillary electrophoresis with laser-induced fluorescence and pre-column derivatization for the analysis of illicit drugs

In the current paper, we report the development of a new capillary electrophoresis method using pre-column derivatization and laser-induced fluorescence detection for the determination of ephedrine and amphetamine drugs. Our new method allows for the identification and quantification of six commonly used illicit drugs namely pseudoephedrine, ephedrine, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, and 3,4-methylenedioxymethylamphetamine, respectively, as well as propafenone (internal standard). Following derivatization with fluorescein isothiocyanate, a total of six amphetamine drugs and the internal standard could readily be separated using a fused-silica 75 micromID x 60 cm length (effective length: 50.2 cm) capillary column. The mobile phase consisted of buffer containing 20mM borate (pH 12, adjusted with sodium hydroxide). Samples were injected in pressure mode with the capillary being operated at 25kV/25 degrees C, and the detection of the derivatized compounds was sought using a laser-induced fluorescence (LIF) detector (lambda(ex)=488 nm and lambda(em)=520 nm), with a run-time of 20 min. The current method was validated with regard to precision (relative standard deviation, RSD), accuracy, sensitivity, linear range, limit of detection (LOD) and limit of quantification (LOQ). In human blood and urine samples, detection limits were 0.2 ngmL(-1), and the linear range of the calibration curves was 0.5-100 ngmL(-1). The intra-day and inter-day precisions were both less than 13.22%.     

Determination of norepinephrine in small volume plasma samples by stable-isotope dilution gas chromatography–tandem mass spectrometry with negative ion chemical ionization

A stable-isotope based gas chromatography–tandem mass spectrometry–negative ion chemical ionization method was developed for the determination of norepinephrine (NE) levels in small volumes (25–100 μl) of plasma. NE was stabilized in plasma by the addition of semicarbazide and spiked with deuterium-labeled norepinephrine internal standard. The analytes were isolated from the plasma by solid-phase extraction using phenylboronic acid columns and derivatized using pentafluoropropionic anhydride. The derivatized analytes were chromatographed on a capillary column and detected by tandem mass spectrometry with negative ion chemical ionization. Unparalleled sensitivity and selectivity were obtained using this detection scheme, allowing the unambiguous analysis of trace levels of NE in small-volume plasma samples. Linear standard curves were obtained for NE over a mass range from 1 to 200 pg per sample. The method had a limit of quantitation of 10 pg NE/ml plasma when using a 100-μl sample aliquot (1 pg/sample). Accuracy for the analysis of plasma samples spiked with 10 to 200 pg NE/ml typically ranged from 100±10%, with RSD values of less than 10%. The methodology was applied to determine the effect of clonidine on plasma NE levels in conscious spontaneously hypertensive rats. Administration of clonidine (30 μg/kg) produced an 80% reduction in plasma NE accompanied by a 30% reduction in heart and mean arterial pressure that persisted >90 min after drug administration. The ability to take multiple samples from individual rats allowed the time course for the effect of clonidine to be mapped out using only one group of animals.     

Liquid chromatographic determination of minocycline in brain-to-plasma distribution studies in the rat

An isocratic reversed-phase high-performance liquid chromatographic procedure was developed for the determination of minocycline in rat plasma and brain and applied to brain-to-blood (plasma) distribution studies. The procedure is based on isolation of the compound and the internal standard (either demeclocycline or tetracycline may be used) from plasma and brain constituents using the Oasis HLB cartridge, with satisfactory recovery and specificity, and separation on a Symmetry Shield RP8 (15 cm x 4.6 mm, 3.5 microm) column coupled with a UV detector set at 350 nm. The assay was linear over a wide range, with a lower limit of quantification of 50 ng ml(-1) or g(-1), using 0.2 ml of plasma and about 200 mg of brain tissue. Precision and accuracy were acceptable. In the rat minocycline crossed the blood-brain barrier slowly, achieving mean brain concentrations between 30 and 40% of the equivalent systemic exposure, regardless of the dose and route of administration.     

Determination of grepafloxacin and clinafloxacin by capillary zone electrophoresis

A simple and rapid capillary zone electrophoresis determination method with UV detection of grepafloxacin and clinafloxacin has been developed. The separation was performed in 35 mM borate-35 mM phosphate buffer solution (pH 8.6), containing 6% (v/v) of acetonitrile. Analyses were realised using fused-silica capillaries (57 cm length x 75 microm I.D.) and the operating conditions were: 15 kV applied voltage, 30 degrees C and detection at 279 nm. Piromidic acid was used as an internal standard. The linear concentration range of application was 1.0-120.0 microg ml(-1) for both compounds, with a detection limit of 0.2 microg ml(-1) for grepafloxacin and 0.3 microg ml(-1) for clinafloxacin. The analysis yielded good reproducibility (RSD between 3.37 and 1.74%). It was applied to the determination of grepafloxacin and clinafloxacin in human and rat urine samples. The method was validated using HPLC as a reference method. Recovery levels were between 94.5 and 103%.