High-performance liquid chromatographic assay for the simultaneous determination of sulfadoxine and pyrimethamine from whole blood dried onto filter paper

A method using solid-phase extraction and high-performance liquid chromatography is evaluated for the simultaneous determination of sulfadoxine and pyrimethamine from 0.1 ml of whole blood dried onto filter paper. Extraction recoveries are about 60% for both drugs. The coefficient of variation for intra-assay precision, inter-assay precision and accuracy is less than 10% for sulfadoxine (10-100 microg/ml) and pyrimethamine (1-10 microg/ml).     

Simultaneous determination of selegiline and desmethylselegiline in human body fluids by headspace solid-phase microextraction and gas chromatography-mass spectrometry

A method for the simultaneous determination of selegiline and its metabolite, desmethylselegiline, in human whole blood and urine is presented. The method, which combines a fiber-based headspace solid-phase microextraction (SPME) technique with gas chromatography-mass spectrometry (GC-MS), required optimization of various parameters (e.g., salt additives, extraction temperatures, extraction times and the extraction properties of the SPME fiber coatings). Pargyline was used as the internal standard. Extraction efficiencies for both selegiline and desmethylselegiline were 2.0-3.4% for whole blood, and 8.0-13.2% for urine. The regression equations for selegiline and desmethylselegiline extracted from whole blood were linear (r(2)=0.996 and 0.995) within the concentration ranges 0.1-10 and 0.2-20 ng/ml, respectively. For urine, the regression equations for selegiline and desmethylselegiline were linear (r(2)=0.999 and 0.998) within the concentration ranges 0.05-5.0 and 0.1-10 ng/ml, respectively. The limit of detection for selegiline and desmethylselegiline was 0.01-0.05 ng/ml for both samples. The lower and upper limits of quantification for each compound were 0.05-0.2 and 5-20 ng/ml, respectively. Intra- and inter-day coefficients of variation for selegiline and desmethylselegiline in both samples were not greater than 8.7 and 11.7%, respectively. The determination of selegiline and desmethylselegiline concentrations in Parkinson's disease patients undergoing continuous selegiline treatment is presented and is shown to validate the present methodology.     

Urinary concentrations of morphine after the administration of herbal teas containing Papaveris fructus in relation to doping analysis

A quantitative method for the analysis of morphine in human urine in the concentration range between 0.25 and 2 microg/ml is described and validated. Morphine was determined after enzymatic hydrolysis of the urine. After liquid-liquid extraction with dichloromethane-methanol (9:1) at pH 9.5, morphine was derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) and analyzed with GC-MS (full scan). The limit of quantification of the method was 0.25 microg/ml. Two Papaveris fructus containing herbal teas were administered to five male volunteers and urine samples were taken quantitatively during the first 12 h after the administration. The morphine concentration in the tea was 10.4 and 31.5 microg/ml, respectively. Morphine was detected in the urine of all volunteers by 1 h after drinking the tea. Maximum morphine concentrations, 4.3 and 7.4 microg/ml, respectively, were obtained 4-6 h after administration. Doping positive urine samples were delivered for 1-9 h.     

Sensitive determination of alpha-methyltryptamine (AMT) and 5-methoxy-N,N-diisopropyltryptamine (5MeO-DIPT) in whole blood and urine using gas chromatography-mass spectrometry

We devised a sensitive and simple method to determine alpha-methyltryptamine (AMT) and 5-methoxy-N,N-diisopropyltryptamine (5MeO-DIPT) in whole blood and urine, using gas chromatography-mass spectrometry (GC-MS). AMT and 5MeO-DIPT were extracted using an Extrelut column with an internal standard, bupivacaine, followed by derivatization with acetic anhydride. The derivatized extract was used for GC-MS analysis of EI-SIM mode. The calibration curves of AMT and 5MeO-DIPT were linear in the concentration range from 10 to 750 ng/ml in both blood and urine samples. The method detection limit (MDL) of AMT and 5MeO-DIPT were 1 ng/ml each in whole blood and 5 ng/ml each in urine. This method should be most useful to accurately determine the presence of these drugs in blood and urine in clinical and forensic cases.     

Direct extract derivatization for determination of amino acids in human urine by gas chromatography and mass spectrometry

The purpose of this study was to develop a simple and accurate analytical method to determine amino acids in urine samples. The developed method involves the employment of an extract derivatization technique together with gas chromatography-mass spectrometry (GC-MS). Urine samples (300 microl) and an internal standard (10 microl) were placed in a screw tube. Ethylchloroformate (50 microl), methanol-pyridine (500 microl, 4:1, v/v) and chloroform (1 ml) were added to the tube. The organic layer (1 microl) was injected to a GC-MS system. In this proposed method, the amino acids in urine were derivatized during an extraction, and the analytes were then injected to GC-MS without an evaporation of the organic solvent extracted. Sample preparation was only required for ca. 5 min. The 15 amino acids (alanine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tyrosine, tryptophan, valine) quantitatively determined in this proposed method. However, threonine, serine, asparagine, glutamine, arginine were not derivatized using any tested derivatizing reagent. The calibration curves showed linearity in the range of 1.0-300 microg/ml for each amino acid in urine. The correlation coefficients of the calibration curves of the tested amino acids were from 0.966 to 0.998. The limit of detection in urine was 0.5 microg/ml except for aspartic acid. This proposed method demonstrated substantial accuracy for detection of normal levels. This proposed method was limited for the determination of 15 amino acids in urine. However, the sample preparation was simple and rapid, and this method is suitable for a routine analysis of amino acids in urine.     

Determination of chromium in urine samples by complexation-supercritical fluid extraction and liquid or gas chromatography

A method was developed for the determination of chromium as the Cr(acac)(3) complex in urine using SFE and chromatography. Quantitative extractions were achieved when the experiments were carried out under 3000 p.s.i. of pressure, at a temperature of 120 degrees C, with 2.0 ml of methanol, 30 min of static extraction and 5 min of dynamic extraction. The chromium was quantified by GC-FID detection. The calibration graph of Cr(acac)(3) solutions was linear between 0.50 and 43.0 microg ml(-1) of chromium (DL 0.18 microg ml(-1), R=0.9994). The same extracts were quantified by HPLC-340 nm detection. The calibration curve of the Cr(acac)(3) solutions was linear over a range of 0.013 to 60.0 microg ml(-1) (DL 0.02 microg ml(-1), R=0.9999). This method was applied to urine samples from 60 diabetic patients and 21 healthy volunteers. Chromium concentration ranges were 2.5-29.5 microg l(-1) for the diabetics and 5.9-12.3 microg l(-1) for the normal subjects.     

Simultaneous determination of bromvalerylurea and allylisopropylacetylurea in human blood and urine by gas chromatography-mass spectrometry

We devised a sensitive and simple method to simultaneously determine bromvalerylurea and allylisopropylacetylurea in human blood and urine by gas chromatography-mass spectrometry. Bromvalerylurea and allylisopropylacetylurea were extracted using an Extrelut column with an internal standard, 2-bromohexanoylurea, followed by derivatization with heptafluorobutyric anhydride. The derivatized extract was submitted to GC-MS analysis of EI-SIM mode. The calibration curves of both compounds were linear in the concentration range from 0.01 to 10 microg/ml in both blood and urine samples. The lower limits of detection of bromvalerylurea and allylisopropylacetylurea were 0.005 and 0.005 microg/ml, respectively. This method proved most useful in accurately identifying these drugs in blood and urine from an autopsied individual.     

Determination of lamotrigine in whole blood with on line solid phase extraction

A simple, sensitive and reproducible method was developed for the determination of lamotrigine in whole blood with on-line solid phase extraction followed by HPLC separation with UV detection. Whole blood samples were diluted 1:1 with water and then injected directly on a clean-up column dry-packed with 40microm C8 silica and separated on a C18 reversed-phase column (150x4.6mm) at room temperature. The extraction column was activated with methanol and conditioned with phosphate buffer of pH 4.5. Mobile phases consisted of phosphate buffer of pH 4.5 for the extraction column and of phosphate buffer of pH 4.5 - acetonitrile (60:40, v/v) for the analytical column. At a flow rate of 1.0ml/min and a connection time of 1.0min, the complete cycle time was 10.0min. Detection was carried out at 260nm. No internal standard was necessary. The method was linear over concentration range 0.2-20.0microg/ml for lamotrigine. Recovery was 98%. Within-day and between-day coefficients of variation ranged from 1.8 to 6.7%.     

Development of simultaneous gas chromatography-mass spectrometric and liquid chromatography-electrospray ionization mass spectrometric determination method for the new designer drugs, N-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl)piperazine (TFMPP) and their main metabolites in urine

To prove the intake of recently controlled designer drugs, N-benzylpiperazine (BZP) and 1-(3-trifluoromethylphenyl)piperazine (TFMPP), a simple, sensitive and reliable method which allows us to simultaneously detect BZP, TFMPP and their major metabolite in human urine has been established by coupling gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). GC-MS accompanied by trifluoroacetyl (TFA) derivatization and LC-MS analyses were performed after the enzymatic hydrolysis and the solid phase extraction with OASIS HLB, and BZP, TFMPP and their major metabolites, 4'-hydroxy-BZP (p-OH-BZP), 3'-hydroxy-BZP (m-OH-BZP) and 4'-hydroxy-TFMPP (p-OH-TFMPP), have found to be satisfactorily separated on a semi-micro SCX column with acetonitrile-40 mM ammonium acetate buffer (pH 4) (75:25, v/v) as the eluent. The detection limits produced by GC-MS were estimated to be from 50 ng/ml to 1 microg/ml in the scan mode, and from 200 to 500 ng/ml in the selected ion monitoring (SIM) mode. Upon applying the LC-ESI-MS technique, the linear calibration curves were obtained by using the SIM mode for all analytes in the concentration range from 10 ng/ml to 10 microg/ml. The detection limits ranged from 5 to 40 ng/ml in the scan mode, and from 0.2 to 1 ng/ml in the SIM mode. These results indicate the high reliability and sensitivity of the present procedure, and this procedure will be applicable for proof of intake of BZP and TFMPP in forensic toxicology.     

Stereospecific determination of mefloquine in biological fluids by high-performance liquid chromatography

A sensitive stereoselective HPLC method was developed for determination of mefloquine (MFQ) enantiomers in plasma, urine and whole blood. The assay involved liquid-liquid extraction of MFQ from biological fluids with a mixture of hexane and isopropanol in the presence of sodium hydroxide and derivatization of the residue by (+)-(S)-naphthylethylisocyanate (NEIC) as chiral derivatizing reagent. Separation of the resulting diastereomers was performed on a silica normal-phase column using chloroform-hexane-methanol (25:74:1) as the mobile phase with a flow-rate of 1 ml/min. Using 200 μl of plasma or whole blood, the limit of determination was 0.2 μg/ml with UV detection for both enantiomers. The limit of determination in 500 μl of urine was 0.08 μg/ml with UV detection.     

Determination of the immunosuppressant mycophenolic acid in human serum by solid-phase microextraction coupled to liquid chromatography

A solid phase microextraction (SPME)-HPLC-UV method for the determination of the immunosuppressant mycophenolic acid (MPA) in human serum samples was developed for the first time. The procedure, that employed a carbowax/templated resin (Carbowax/TPR-100) as fiber coating, required a very simple sample pretreatment, an isocratic elution, and provides an highly selective extraction. The linear range was 0.2-100 microg x ml(-1). Recovery was practically unchanged (63+/- 4%) passing from 0.2 to 100 microg x ml(-1) level. Within-day and between-days coefficient of variation ranged from 5.9 to 6.5% and from 8.8 to 9.2%, respectively. A detection limit of 0.05 microg x ml(-1) was estimated in spiked serum. The method was successfully applied to the determination of MPA in serum of a patient under mycophenolate mophetil ester (MMF) therapy, as demonstrated by the relevant concentration-time profiles.     

Ultrasonic-assisted derivatization reaction of amino acids prior to their determination in urine by using single-drop microextraction in conjunction with gas chromatography

A derivatization-extraction method that avoids tedious preconcentration steps is established in order to determine amino acids accurately at nanogram levels. The method involves conversion of the analytes of concern to N(O,S)-ethoxycarbonyl amino acid ethyl esters and subsequent extraction by single-drop microextraction (SDME) followed by GC analysis. The reaction proceeds smoothly and rapidly under ultrasonication which removes the bubbles from the bulk solution. Precision is acceptable and 12 non-hydrolyzed amino acids can be determined in urine in this manner. As long as the extraction conditions are consistently applied, quantitative analysis can be performed accurately. The limits of detection were satisfactory in the range 0.010-0.025 microg/ml for GC-FID and 0.26-68 ng/ml for GC-MS(SIM) with 1 ml sample volume.     

Liquid chromatography-electrospray mass spectrometry determination of a bis-thiazolium compound with potent antimalarial activity and its neutral bioprecursor in human plasma, whole blood and red blood cells

Liquid chromatography-electrospray ionization mass spectrometry methods are described for the simultaneous quantification of a bis-thiazolium compound (T3), its related prodrug (TE3) and an intermediate compound (mTE3) that appeared during the prodrug/drug conversion process, in human plasma, whole blood and red blood cells (RBCs). The methods involve solid phase extraction (SPE) of the compounds and the internal standard (verapamil) from the three different matrices using OasisHLB columns with an elution solvent of 2x1 ml of acetonitrile containing 1 ml/l trifluoroacetic acid (TFA). HPLC separation was performed on a C18 encapped Xterra column packed with 3.5 microm particles. The mobile phase used a 8 min gradient, from water containing 1 ml/l TFA to acetonitrile containing 1 ml/l TFA, at a flow rate of 400 microl/min. Verapamil and the TE3 compound were characterized by the protonated molecules at m/z 455 and m/z 541, respectively. The mTE3 species was detected through the (M)+ ion at m/z 497. The T3 compound was detected by use of two ions, the quaternary ammonium salt (M2+/2) at m/z 227.3 and by the adduct with TFA (M+TFA)+ at m/z 567.3. The drug/internal standard peak area ratios were linked via a quadratic relationship to plasma (or whole blood) concentrations in the tested range of 6.4-1282 microg/l (12.8-2564 microg/kg) for T3, 20-2000 microg/l (40-4000 microg/kg) for mTE3 and 10-2000 microg/l (40-4000 microg/kg) for TE3, and to T3 concentrations in RBCs ranging from 12.8 to 2564 microg/kg. Inter-assay precision (in terms of R.S.D.) was below 13.5% and accuracy ranged from 95.4 to 107%. The dilution of the samples (plasma or whole blood) has no influence on the performance of the methods. The extraction recoveries averaged 87% for T3, 53% for mTE3 and 79% for TE3 in plasma; 79% for T3, 57% for mTE3 and 65% for TE3 in blood; and 93% for T3 in RBCs, and was constant across the calibration range. The lower limits of quantitation were 6.4 microg/l for T3, 20 microg/l for mTE3 and 10 microg/l for TE3 in plasma; 12.8 microg/kg for T3 and 40 microg/kg for mTE3 and TE3 in blood; and 12.8 microg/kg for T3 in RBCs. Stability tests under various conditions were also investigated. The three-step SPE procedure (loading, clean-up, and elution) described in this paper to quantify these new anti-malarial compounds in plasma, whole blood and RBCs, can easily be automated by using either robotisation or an automated sample preparation system.     

Determination of quinalphos in blood and urine by direct solid-phase microextraction combined with gas chromatography-mass spectrometry

A new method based on direct solid-phase microextraction (DI-SPME) followed by gas chromatography-mass spectrometry was developed for the purpose of determining quinalphos in blood and urine. Two types of coated fibre have been assayed and compared: carbowax/divinylbenzene (CW/DVB 65 microm) and polydimethylsiloxane (PDMS 100 microm). The main parameters affecting the SPME process such as temperature, salt addition, pH, stirring and adsorption/desorption time profiles were optimized to enhance the sensitivity of the procedure. The method was developed using only 100 microL of blood and urine. Limits of detection of the method for blood and urine matrices were, respectively, 10 and 2 ng/mL. Linearity was established over concentration ranges from 0.05 to 50 microg/mL for blood, and 0.01 to 50 microg/mL for urine, with regression coefficients ranging between 0.9991 and 0.9999. Intra- and interday precision values were less than 13%, and accuracy was within +/-15% of the nominal concentration for all studied levels in both matrices. Absolute recoveries were 14 and 26% for blood and urine, respectively.     

Standardization of the method for estimation of ethambutol in pharmaceutical preparations and biological fluid

A simple column chromatographic method for determination of ethambutol (EMB) in pharmaceutical preparations containing EMB in combination with other anti-TB drugs is presented. The method involved extraction of EMB into an organic solvent, followed by basification and column chromatographic separation on Amberlite CG 50 (100-200 mesh) and elution with suitable eluants and estimation at a wavelength of 270 nm. The assay was linear from 25 to 400 microg/ml. The relative standard deviations of intra and inter day assays were lower than 5%. Ethambutol was recovered from human urine quantitatively and stable for a period of at least one week in urine stored at -20 degrees C.     

Liquid chromatography-electrospray mass spectrometry determination of ibogaine and noribogaine in human plasma and whole blood. Application to a poisoning involving Tabernanthe iboga root

A liquid chromatography/electrospray ionization mass spectrometry (LC-ESI-MS) method was developed for the first time for the determination of ibogaine and noribogaine in human plasma and whole blood. The method involved solid phase extraction of the compounds and the internal standard (fluorescein) from the two matrices using OasisHLB columns. LC separation was performed on a Zorbax eclipse XD8 C8 column (5 microm) with a mobile phase of acetonitrile containing 0.02% (v/v) trimethylamine and 2mM ammonium formate buffer. MS data were acquired in single ion monitoring mode at m/z 311.2, 297.2 and 332.5 for ibogaine, noribogaine and fluorescein, respectively. The drug/internal standard peak area ratios were linked via a quadratic relationship to plasma (0.89-179 microg/l for ibogaine; 1-200 microg/l for noribogaine) and to whole blood concentrations (1.78-358 microg/kg for ibogaine; 2-400 microg/kg for noribogaine). Precision ranged from 4.5 to 13% and accuracy was 89-102%. Dilution of the samples had no influence on the performance of the method. Extraction recoveries were > or =94% in plasma and > or =57% in whole blood. The lower limits of quantitation were 0.89 microg/l for ibogaine and 1 microg/l for noribogaine in plasma, and 1.78 microg/kg for ibogaine and 2 microg/kg for noribogaine in whole blood. In frozen plasma samples, the two drugs were stable for at least 1 year. In blood, ibogaine and noribogaine were stable for 4h at 4 degrees C and 20 degrees C and 2 months at -20 degrees C. The method was successfully used for the analysis of a poisoning involving Tabernanthe iboga root.     

Determination of dichloromethane, trichloroethylene and perchloroethylene in urine samples by headspace solid phase microextraction gas chromatography-mass spectrometry

A method for the determination of volatile chlorinated hydrocarbons, namely dichloromethane (DCM), trichloroethylene (TCE), and perchloroethylene (PCE), in urine samples was developed using headspace solid phase microextraction (HS-SPME) gas chromatography-mass spectrometry (GC-MS). HS-SPME was performed using a 75 microm Carboxen-polydimethylsiloxane fiber. Factors, which affect the HS-SPME process, such as adsorption and desorption times, stirring, salting-out effect, and temperature of sampling have been evaluated and optimized. The highest extraction efficiency was obtained when sampling was performed at room temperature (22 degrees C), from samples saturated with salt and under agitation. Linearity of the HS-SPME-GC-MS method was established over four orders of magnitude and the limit of detection was 0.005 microg/l for all the compounds. Precision, calculated as %R.S.D. at three different concentration levels, was within 1-8% for all intra- and inter-day determinations. The method was applied to the quantitative determination of TCE and PCE in human urine samples from exposed (TCE, n=5; median, 9.32 microg/l and PCE, n=39; median, 0.58 microg/l) and non-exposed individuals (n=120; median concentrations, 0.64, 0.22 and 0.11 microg/l for DCM, TCE and PCE, respectively. In addition, two cases of acute accidental exposure to DCM are reported, and the elimination kinetics in blood and urine was followed up. The calculated half-lives of urinary and blood DCM were, respectively, 7.5 and 8.1 h for one subject and 3.8 and 4.3 h for the other.     

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%.     

Determination of phenylbutazone and oxyphenbutazone in plasma and urine samples of horses by high-performance liquid chromatography and gas chromatography-mass spectrometry

A method is described for the qualiitative and quantitative determination of phenylbutazone and oxyphenbutazone in horse urine and plasma samples viewing antidoping control. A horse was administered intravenously with 3 g of phenylbutazone. For the qualitative determination, a screening by HPLC was performed after acidic extraction of the urine samples and the confirmation process was realized by GC-MS. Using the proposed method it was possible to detect phenylbutazone and oxyphenbutazone in urine for up to 48 and 120 h, respectively. For the quantitation of these drugs the plasma was deproteinized with acetonitrile and 20 gml were injected directly into the HPLC system equipped with a UV detector and LiChrospher RP-18 column. The mobile phase used was 0.01 M acetic acid in methanol (45:55, v/v). The limit of detection was 0.5 μg/ml for phenylbutazone and oxyphenbutazone and the limit of quantitation was 1.0 μg/ml for both drugs. Using the proposed method it was possible to quantify phenylbutazone up to 30 h and oxyphenbutazone up to 39 h after administration.     

Simultaneous determination of three residual barbiturates in pork using accelerated solvent extraction and gas chromatography-mass spectrometry

A new method was developed for the rapid extraction and unequivocal determination of barbital, amobarbital and phenobarbital residues in pork. The isolation of the analytes from pork samples was accomplished by utilizing an accelerated solvent extractor ASE 300. The procedure was automatically carried out in series for fat removing and extraction, respectively with n-hexane and acetonitrile pressurized constantly at 10.3 MPa for 30 min. After evaporation, the extracts were cleaned up on a C(18) solid phase extraction (SPE) cartridge and the barbiturates were eluted with hexane-ethyl acetate (7:3), evaporated on a rotary evaporator and derivatized with CH(3)I. The methylated barbiturates were separated on a HP-5MS capillary column and detected with a mass detector. Electron impact ion source (EI) operating in time program-selected ion monitoring mode (SIM) was used for identification and external standard method was used for quantification. Good linearity was obtained in the range from 0.5 microg/kg to 25 microg/kg. Average recoveries of the three barbiturates spiked in pork ranged from 84.0% to 103.0%, with relative standard deviations from 1.6% to 12%. The limit of detection (LOD) was 0.5 microg/kg for the three barbiturates (S/N>or=3). The quantification limit (LOQ) was 1 microg/kg for the three barbiturates (S/N>or=10).