Validation and implementation of a liquid chromatography/tandem mass spectrometry assay to quantitate dimethyl benzoylphenylurea (BPU) and its five metabolites in human plasma and urine for clinical pharmacology studies

A method has been developed for the quantitation of N-[4-(5-bromo-2-pyrimidinyloxy)-3-methylphenyl]-N'-(2-dimethylamino-benzoyl)urea (BPU) and its metabolites in human plasma and urine. BPU and metabolites were separated on a C18 column with acetonitrile-water mobile phase containing 0.1% formic acid using isocratic flow for 5 min. The analytes were monitored by tandem mass spectrometry. Calibration curves were generated over the range of 2.5-500 ng/mL for BPU, mmBPU, and aminoBPU in plasma; and 0.1-20, 0.1-20, 0.5-100, 10-2000, 1-200, and 3-600 ng/mL for BPU, mmBPU, aminoBPU, G280, G308, and G322 in urine, respectively. The method has been successfully applied to study the pharmacokinetics of BPU.     

A rapid and accurate UPLC/MS/MS method for the determination of benzodiazepines in human urine

A rapid, sensitive, and specific ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) assay method for simultaneous determination of 13 benzodiazepine compounds in human urine was developed and validated. Aliquots of 0.5 mL of urine specimens were used for the analysis and the benzodiazepines were extracted by single step methanol (containing 0.2% formic acid) precipitation and then separated on a BEH C18 (50 mm × 2.1 mm, 1.7 μm) analytical column with the temperature maintained at 45°C. The mobile phases consisted of methanol and water (both containing 0.2% formic acid) and the flow rate was 0.4 mL/min. The TQ detector, equipped with an electrospray ionization ion source, was set up with a positive mode. The acquisitions were performed in multiple-reaction monitoring (MRM) and the limit of quantification was 20 ng/mL for all of the 13 compounds. The low limits of detections (LODs) of the benzodiazepines in this method were between 0.5 and 2 ng/mL. The chromatographic separation time was 4 min and calibration curves in human urine were generated over the range of 20-2000 ng/mL. The method validation parameters such as accuracy, precision, carryover, recovery, stability, and specificity for all of the 13 compounds were within the acceptable range. This method is suitable for the high throughput screening of benzodiazepines in clinical laboratories.     

A sensitive liquid chromatography-mass spectrometry method for simultaneous determination of two active chromones from Saposhnikovia root in rat plasma and urine

A sensitive and efficient liquid chromatography-mass spectrometry method was developed and validated for the simultaneous determination of two active chromones (prim-O-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol) from Saposhnikovia root in rat plasma and urine. The plasma or urine samples were prepared by protein precipitation. Chromatographic separation of the two active chromones from matrix interferences was achieved on an Angilent TC-C(18) column with a mobile phase consisted of methanol, water and 0.1% formic acid. Puerarin was added as the internal standard. The method was validated with the concentration range 1.0-100 ng/mL in rat plasma and 10-1000 ng/mL in urine for prim-O-glucosylcimifugin, 1.5-150 ng/mL in plasma and 15-1500 ng/mL in urine for 4'-O-D-glucosyl-5-O-methylvisamminol. The lower limit of quantitation (LLOQ) of prim-O-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol was 1.0 and 1.5 ng/mL in plasma, 10 and 15 ng/mL in urine, respectively. The intra- and inter-day precision across three validation days over the entire concentration range was lower than 9.0% as terms of relative standard deviation (R.S.D.). Accuracy determined at three quality control concentrations (2.0, 25 and 75 ng/mL for prim-O-glucosylcimifugin; 3.0, 37.5 and 112.5 ng/mL for 4'-O-D-glucosyl-5-O-methylvisamminol) ranged from -1.9 to 3.9% as terms of relative error (R.E.). The LC-ESI-MS method was further applied to assess pharmacokinetics and urine excretion of the two chromones after oral administration of Fangfeng extract to rats. Practical utility of this new LC-MS method was confirmed in pilot pharmacokinetic studies in rats following oral administration.     

Quantification of sunitinib in human plasma by high-performance liquid chromatography-tandem mass spectrometry

A rapid, sensitive and specific method was developed and validated using LC/MS/MS for determination of sunitinib in human plasma. Sample preparation involved a liquid-liquid extraction by the addition of 0.2mL of plasma with 4.0mL tert-butyl-methyl-ether extraction solution containing 25ng/mL of the internal standard clozapine. Separation of compounds was achieved on a C18 (50mmx2.1mm i.d., 3.5microm) analytical column using a mobile phase consisting of acetonitrile/H20 (65:35, v/v) containing 0.1% formic acid and isocratic flow at 0.150mL/min for 3min. The analytes were monitored by tandem-mass spectrometry with electrospray positive ionization. Linear calibration curves in human plasma were generated over the range of 0.2-500ng/mL with values for the coefficient of determination of >0.9950. Within- and between day precision and accuracy were < or =10%. The method was applied to the quantitation of sunitinib in plasma samples from a patient receiving daily oral therapy with sunitinib.     

A dried blood spots technique based LC-MS/MS method for the analysis of posaconazole in human whole blood samples

A rugged and robust liquid chromatographic tandem mass spectrometric (LC-MS/MS) method utilizing dried blood spots (DBS) was developed and validated for the analysis of posaconazole in human whole blood. Posaconazole fortified blood samples were spotted (15 μL) onto Ahlstrom Alh-226 DBS cards and dried for at least 2h. Punched spots were then extracted by using a mixture of acetonitrile and water containing stable labeled internal standard (IS). Posaconazole and its IS were separated from endogenous matrix components on a Kinetex? C18 column under gradient conditions with a mobile phase A consisting of 0.1% formic acid and a mobile phase B consisting of 0.1% formic acid in acetonitrile/methanol (70/30, v/v). The analyte and IS were detected using a Sciex API 4000 triple quadrupole LC-MS/MS system equipped with a TurboIonSpray? source operated in the positive ion mode. The assay was linear over the concentration range of 5-5000 ng/mL. The inter-run accuracy and precision of the assay were -1.8% to 0.8% and 4.0% to 10.4%, respectively. Additional assessments unique to DBS were investigated including sample spot homogeneity, spot volume, and hematocrit. Blood spot homogeneity was maintained and accurate and precise quantitation results were obtained when using a blood spot volume of between 15 and 35 μL. Human blood samples with hematocrit values ranging between 25% and 41% gave acceptable quantitation results. The validation results indicate that the method is accurate, precise, sensitive, selective and reproducible.     

固相萃取-高效液相色谱法测定复合薯片中丙烯酰胺

建立了一种利用固相萃取小柱与高效液相色谱联用,采用光电二极管阵列检测器测定复合薯片中丙烯酰胺的方法。以0．1％的甲酸水溶液为提取试剂,采用Waters Oasis HLB固相萃取小柱（200mg／6cc）对提取液净化处理,然后以流动相为甲醇／水（5：95,V：V）,流速为0．6mL／min,检测波长为210nm,利用AgilentC18反相色谱柱进样分析。在该条件下丙烯酰胺的回收率达80％以上,最低检测限小于10ng／mL。该方法操作简便、重复性好、稳定性高,可用于油炸复合薯片中丙烯酰胺的快速检测。     

Specific method for determination of gefitinib in human plasma, mouse plasma and tissues using high performance liquid chromatography coupled to tandem mass spectrometry

A rapid, sensitive and specific method was developed and validated using liquid chromatography-tandem mass spectrometry (LC/MS/MS) for determination of gefitinib in human plasma and mouse plasma and tissue. Sample preparation involved a single protein precipitation step by the addition of 0.1 mL of plasma or a 200 mg/mL tissue homogenate diluted 1/10 in human plasma with 0.3 mL acetonitrile. Separation of the compounds of interest, including the internal standard (d8)-gefitinib, was achieved on a Waters X-Terra C18 (50 mm x 2.1 mm i.d., 3.5 microm) analytical column using a mobile phase consisting of acetonitrile-water (70:30, v/v) containing 0.1% formic acid and isocratic flow at 0.15 mL/min for 3 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 1-1000 ng/mL for the human plasma samples and 5-1000 ng/mL for mouse plasma and tissue samples with values for the coefficient of determination of > 0.99. The values for both within- and between-day precision and accuracy were well within the generally accepted criteria for analytical methods (< 15%). This method was subsequently used to measure concentrations of gefitinib in mice following administration of a single dose of 150 mg/kg intraperitoneally and in cancer patients receiving an oral daily dose of 250 mg.     

Pharmacokinetics of ergosterol in rats using rapid resolution liquid chromatography-atmospheric pressure chemical ionization multi-stage tandem mass spectrometry and rapid resolution liquid chromatography/tandem mass spectrometry

Rapid resolution liquid chromatography/tandem multi-stage mass spectrometry (RRLC-MS(n)) and rapid resolution liquid chromatography/tandem mass spectrometry (RRLC/MS/MS) methods were developed for the identification and quantification of ergosterol and its metabolites from rat plasma, urine and faeces. Two metabolites (ERG1 and ERG2) were identified by RRLC/MS(n). The concentrations of the ergosterol were determined by RRLC/MS/MS. The separation was performed on an Agilent Zorbax SB-C18 with the mobile phase consisting of methanol and water (containing 0.1% formic acid). The detection was carried out by means of atmospheric pressure chemical ionization mass spectrometry in positive ion mode with multiple reaction monitoring (MRM). Linear calibration curves were obtained in the concentration range of 7-2000, 6-2000 and 8-7500 ng/mL for plasma, urine and faecal homogenate, respectively. The intra- and inter-day precision values (RSD) were below 10%. The method was applied to the pharmacokinetic properties and elimination pathway of ergosterol in rats.     

Liquid chromatography/tandem mass spectrometry method for simultaneous evaluation of activities of five cytochrome P450s using a five-drug cocktail and application to cytochrome P450 phenotyping studies in rats

A reliable liquid chromatography/tandem mass spectrometry has been developed for simultaneous evaluation of the activities of five cytochrome P450s (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A) in rat plasma and urine. The five-specific probe substrates/metabolites include phenacetin/paracetamol (CYP1A2), tolbutamide/4-hydroxytolbutamide and carboxytolbutamide (CYP2C9), mephenytoin/4'-hydroxymephenytoin (CYP2C19), dextromethorphan/dextrorphan (CYP2D6), and midazolam/1'-hydroxymidazolam (CYP3A). Internal standards were brodimoprim (for phenacetin, paracetamol, midazolam and 1'-hydroxymidazolam), ofloxacin (for 4'-hydroxymephenytoin, dextromethorphan and dextrorphan) and meloxicam (for tolbutamide, 4-hydroxytolbutamide and carboxytolbutamide). Sample preparation was conducted with solid-phase extraction using Oasis HLB cartridges. The chromatography was performed using a C(18) column with mobile phase consisting of methanol/0.1% formic acid in 20 mM ammonium formate (75:25). The triple-quadrupole mass spectrometric detection was operated in both positive mode (for phenacetin, paracetamol, midazolam, 1'-hydroxymidazolam, brodimoprim, 4'-hydroxymephenytoin, dextromethorphan, dextrorphan and ofloxacin) and negative mode (for tolbutamide, 4-hydroxytolbutamide, carboxytolbutamide and meloxicam). Multiple reaction monitoring mode was used for data acquisition. Calibration ranges in plasma were 2.5-2500 ng/mL for phenacetin, 2.5-2500 ng/mL for paracetamol, 5-500 ng/mL for midazolam, and 0.5-500 ng/mL for 1'-hydroxymidazolam. In urine calibration ranges were 5-1000 ng/mL for dextromethorphan, 0.05-10 microg/mL for dextrorphan and 4'-hydroxymephenytoin, 5-2000 ng/mL for tolbutamide, 0.05-20 microg/mL for 4-hydroxytolbutamide and 0.025-10 microg/mL for carboxytolbutamide. The intra- and inter-day precision were 4.3-12.4% and 1.5-14.8%, respectively for all of the above analytes. The intra- and inter-day accuracy ranged from -9.1 to 8.3% and -10 to 9.2%, respectively for all of the above analytes. The lower limits of quantification were 2.5 ng/mL for phenacetin and paracetamol, 5 ng/mL for midazolam, 0.5 ng/mL for 1'-hydroxymidazolam, 5 ng/mL for dextromethorphan, 50 ng/mL for dextrorphan and 4'-hydroxymephenytoin, 5 ng/mL for tolbutamide, 50 ng/mL for 4-hydroxytolbutamide and 25 ng/mL for carboxytolbutamide. All the analytes were evaluated for short-term (24 h, room temperature), long-term (3 months, -20 degrees C), three freeze-thaw cycles and autosampler (24 h, 4 degrees C) stability. The stability of urine samples was also prepared with and without beta-glucuronidase incubation (37 degrees C) and measured comparatively. No significant loss of the analytes was observed at any of the investigated conditions. The current method provides a robust and reliable analytical tool for the above five-probe drug cocktail, and has been successfully verified with known CYP inducers.     

Determination of higenamine in human plasma and urine using liquid chromatography coupled to positive electrospray ionization tandem mass spectrometry

Higenamine is an active ingredient of Aconite root in Chinese herbal medicine and might be used as a new agent for a pharmaceutical stress test and was approved to undergo clinical pharmacokinetic study. Therefore, there exists a need to establish a sensitive and rapid method for the determination of higenamine in human plasma and urine. This paper described a sensitive and rapid method based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for the determination of higenamine in human plasma and urine. Solid-phase extraction (SPE) was used to isolate the compounds from biological matrices followed by injection of the extracts onto an Atlantis dC18 column with isocratic elution. The mobile phase was 0.05% formic acid in water-methanol (40:60, v/v). The mass spectrometry was carried out using positive electrospray ionization (ESI) and data acquisition was carried out in the multiple reaction monitoring (MRM) mode. The method was fully validated over the concentration range of 0.100-50.0 ng/mL and 1.00-500 ng/mL in plasma and urine, respectively. The lower limits of quantification (LLOQs) were 0.100 and 1.00 ng/mL in plasma and urine, respectively. Inter- and intra-batch precision was less than 15% and the accuracy was within 85-115% for both plasma and urine. Extraction recovery was 82.1% and 56.6% in plasma and urine, respectively. Selectivity, matrix effects and stability were also validated in human plasma and urine. The method was applied to the pharmacokinetic study of higenamine hydrochloride in Chinese healthy subjects.     

Analysis of methanol or formic acid in body fluids by headspace solid-phase microextraction and capillary gas chromatography

Methanol and its metabolite formic acid have been found extractable from human whole blood and urine by headspace solid-phase microextraction (SPME) with a Carboxen/polydimethylsiloxane fiber. The headspace SPME for formic acid was carried out after derivatization to methyl formate under acidic conditions. The determinations of both compounds were made by using acetonitrile as internal standard (IS) and capillary gas chromatography (GC) with flame ionization detection. The headspace SPME–GC gave sharp peaks for methanol, methyl formate and I.S.; and low background noises for whole blood and urine samples. Extraction efficiencies were 0.25–1.05% of methanol and 0.38–0.84% formic acid for whole blood and urine. The calibration curves for methanol and formic acid showed excellent linearity in the range of 1.56 to 800 and 1.56 to 500 μg/0.5 ml of whole blood or urine, respectively. The detection limits were 0.1–0.5 μg/0.5 ml for methanol and 0.6 μg/0.5 ml for formic acid for both body fluids. The within-day relative standard deviations in terms of extraction efficiency for both compounds in whole blood and urine samples were not greater than 9.8%. By using the established SPME method, methanol and formic acid were successfully separated and determined in rat blood after oral administration of methanol.     

A rapid and sensitive method for determination of sorafenib in human plasma using a liquid chromatography/tandem mass spectrometry assay

A rapid, sensitive and specific method was developed and validated using LC/MS/MS for determination of sorafenib in human plasma. Sample preparation involved a single protein precipitation step by the addition of 0.1 mL of plasma with 0.5 mL acetonitrile. Analysis of the compounds of interest including the internal standard ([(2)H(3)(15)N] sorafenib) was achieved on a Waters X-Terra C(18) (150 mm x 2.1mm i.d., 3.5 microm) analytical column using a mobile phase consisting of acetonitrile/10 mM ammonium acetate (65:35, v/v) containing 0.1% formic acid and isocratic flow at 0.2 mL/min for 6 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 7.3-7260 ng/mL for the human plasma samples with values for the coefficient of determination of >0.96. The values for both within day and between day precision and accuracy were well within the generally accepted criteria for analytical methods (<15%).     

Simultaneous UFLC-ESI-MS/MS determination of piperine and piperlonguminine in rat plasma after oral administration of alkaloids from Piper longum L.: application to pharmacokinetic studies in rats

The alkaloids from Piper longum L. showed protective effects on Parkinson's disease models in our previous study and piperine and piperlonguminine were the two main constituents in the alkaloids. The present study aimed at developing a rapid, sensitive, and accurate UFLC-ESI-MS/MS method and validating it for the simultaneous determination of piperine and piperlonguminine in rat plasma using terfenadine as the internal standard. The analytes and internal standard (IS) were extracted from rat plasma using a simple protein precipitation by adding methanol/acetonitrile (1:1, v/v). A Phenomenex Gemini 3 u C18 column (20 mm × 2.00 mm, 3 μm) was used to separate the analytes and IS using a gradient mode system with a mobile phase consisting of water with 0.1% formic acid (mobile phase A) and acetonitrile with 0.1% formic acid (mobile phase B) at a flow rate of 0.4 mL/min and an operating column temperature of 25°C. The total analytical run time was 4 min. The detection was performed using the positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode with transitions at m/z 286.1-201.1 for piperine, m/z 274.0-201.1 for piperlonguminine, and m/z 472.4-436.4 for the IS. The calibration curves were both linear (r>0.995) over a concentration range of 1.0 to 1000 ng/mL; the lower limit of quantification (LLOQ) was 1.0 ng/mL for both piperine and piperlonguminine. The intra-day and inter-day precisions (RSD %) were <12.1%, accuracies ranged from 86.6 to 120%, and recoveries ranged from 90.4 to 108%. The analytes were proven stable in the short-term, long-term, and after three freeze-thaw cycles. The method was successfully applied to pharmacokinetic studies of piperine and piperlonguminine in rats after oral administration of alkaloids from P. longum L.     

Determination of PF-04928473 in human plasma using liquid chromatography with tandem mass spectrometry

A simple, rapid and sensitive liquid chromatography/tandem mass spectrometric (LC/MS/MS) analytical method was developed for quantification of Hsp90 inhibitor PF-04928473 in human plasma, following administration of its prodrug, PF-04929113. Sample processing involved protein precipitation by addition of 0.4 mL of methanol containing internal standard (PF-04972487) to 50 μL volume of plasma sample. Chromatographic separation of PF-04928473 and PF-04972487 was achieved on a Phenomenex^® Luna C18(2) (2.0mm × 50 mm, 5 μm) column using a gradient elution method with mobile phase solvents: methanol containing 0.1% formic acid and 0.1% formic acid at a flow rate of 0.25 mL/min. Detection was performed in electrospray positive ionization mode, monitoring the ion transitions from m/z 465.1 → 350.1 (PF-04928473) and m/z 447.0 → 329.1 (PF-04972487). The retention times for PF-04928473 and PF-04972487 were 1.86 and 2.85 min, respectively. Calibration curves were generated in the range of 2–2000 ng/mL. The accuracy and precision ranged from 94.1 to 99.0% and 86.7 to 97.6%, respectively, which were calculated using quality control samples of three different concentrations analyzed in quintuplicate on four different days.     

Simultaneous determination of ribavirin and ribavirin base in monkey plasma by high performance liquid chromatography with tandem mass spectrometry

For the first time, a liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the simultaneous determination of ribavirin and rabavirin base was developed and validated over the concentration range of 10-5,000 ng/ml, respectively, using a 0.025 ml monkey plasma sample. Ribavirin, ribavirin base, and the internal standards were extracted from monkey plasma via protein precipitation. After evaporation of the supernatant, the extract was reconstituted with 5% methanol (containing 0.1% formic acid) and injected onto the LC-MS/MS system. Optimum chromatographic separation was achieved on a Waters Atlantis dc18 (150 mm x 2.1mm, 5 microm) column with mobile phase run in gradient with 100% water containing 0.5% formic acid (A) and 90% acetonitrile (containing 0.5% formic acid (B). The flow rate was 0.4-0.6 ml/min with total cycle time of approximately 7.0 min. Post-column addition of acetonitrile (containing 0.1% formic acid) at 0.3 ml/min was used to increase the ionization efficiency in the MS source. The method was validated for sensitivity, linearity, reproducibility, stability and recovery. Lack of adverse matrix effect and carry-over was also demonstrated. The intra-day and inter-day precision and accuracy of the quality control (QC) samples were <9.0% relative standard deviation (R.S.D.) and 10.8% bias for ribavirin, and 10.3% R.S.D. and 11.3% bias for ribavirin base. The current specific, accurate and precise assay is useful in support of the toxicokinetic and pharmacokinetic studies of these compounds.     

LC-MS/MS method for simultaneous determination of valproic acid and major metabolites in human plasma

A rapid and sensitive method using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) was developed and validated for simultaneous quantitative determination of valproic acid and three major metabolites (3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid) in human plasma. The analytes and internal standard were isolated from 200 μL samples by solid phase extraction using a ZORBAX SB-C? column (3.5 μm, 2.1×100 mm) with an isocratic mobile phase consisting of methanol-10mM ammonium acetate (80:20, v/v) containing 0.1% formic acid at a flow rate of 0.3 mL/min. The method had a chromatographic total run time of 2.0 min. The lower limit of quantification of valproic acid, 3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid of the method was 2030, 51.5, 50.15 and 51.25 ng/mL, respectively. The method was linear for valproic acid and the three metabolites with correlation coefficients >0.995 for all analytes. The intra-day and inter-day accuracy and precision of the assay were less than 15.0%. This analytical method was successfully used to assay plasma concentrations of valproic acid and the three metabolites in human plasma from epileptic patients.     

An LC-MS/MS assay to determine plasma pharmacokinetics of cyclic thymic hexapeptide (cTP6) in rhesus monkeys

A robust and simple method for absolute quantification of a novel bidirectional immunomodulatory drug candidate, cyclic thymic hexapeptide (cTP6), in rhesus monkey plasma was developed and validated by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Plasma proteins were precipitated by adding four volumes of acetonitrile. Peptides in the supernatant were separated by liquid chromatography on an Agilent Zorbax Eclipse Plus-C18 chromatographic column with gradient elution using 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in methanol (mobile phase B) at 0.2 mL/min. The analytes were identified by triple quadrupole mass spectrometry in positive ion-mode. The assay was linear over a concentration range of 10-5000 ng/mL for cTP6, with a lower limit of quantification (LLOQ) of 10 ng/mL. Intra- and inter-day precision of the assay at three concentrations were 1.51-7.70% with accuracy of 95.1-104.2%. The average recovery of cTP6 for three concentration levels was 59.6-64.0%. No significant matrix effect was observed. Peptide cTP6 was detected in plasma of live rhesus monkeys up to 6-8h after intra-muscular injection. The half-life was 2.24-2.95 h. The result revealed a nonlinear pharmacokinetic response to increasing doses of cTP6 (100, 200, 500 μg/kg). For the multiple dose study of cTP6, the drug did not accumulate during daily administration at 100 μg/kg for 7 consecutive days in rhesus monkeys.     

Liquid chromatography-tandem mass spectroscopy assay for quercetin and conjugated quercetin metabolites in human plasma and urine

A sensitive and specific method was developed and validated for the quantitation of quercetin in human plasma and urine. The application of liquid chromatography-tandem mass spectrometry (LC/MS/MS) with a TurboIonspray (TIS) interface in negative mode under multiple reactions monitoring was investigated. Chromatographic separation was achieved on a C12 column using a mobile phase of acetonitrile/water with 0.2% formic acid (pH 2.4) (40/60, v/v). The detection limit was 100 pg/ml and the lower limit of quantification was 500 pg/ml for plasma samples; the detection limit was 500 pg/ml and the lower limit of quantification was 1 ng/ml for urine samples. The calibration curve was linear from 1 to 800 ng/ml for plasma samples and was linear from 1 to 200 and 50 to 2000 ng/ml for urine samples. All the intra- and inter-day coefficients of variation were less than 11% and intra- and inter-day accuracies were within +/-15% of the known concentrations. This represents a LC/MS/MS assay with the sensitivity and specificity necessary to determine quercetin in human plasma and urine. This assay was used to determine both parent quercetin and the quercetin after enzymatic hydrolysis with beta-glucuronidase/sulfatase in human plasma and urine samples following the ingestion of quercetin 500 mg capsules.     

Determination of cycloserine in human plasma by high-performance liquid chromatography with fluorescence detection,using derivatization with p-benzoquinone

A new method for determining cycloserine in plasma samples is described. This method is based on the derivatization of cycloserine with p-benzoquinone, a reaction that takes place at the same time as the process of plasma deproteinization due to the presence of ethanol as solvent in the solution of the derivatization reagent. Four derivatives are obtained from this reaction. The main derivative is well correlated with the cycloserine concentration. The ratio between the volumes of the plasma sample and the reagent solution is 1:2 for a p-benzoquinone concentration of 1000 μg/mL. Elution from a C₁₈ column was isocratic, using a mobile phase containing (v/v) 85% aqueous 0.1% formic acid solution, and 15% (v/v) of a mixture of methanol and acetonitrile (1:1), with a flow-rate of 1 mL/min, at 25°C. Determinations by fluorescence detection were achieved with excitation at 381 nm and emission at 450 nm, with a detection limit of 10 ng/mL for an injection volume of 5 μL. This method was validated and applied to the determination of cycloserine in blood plasma samples of several healthy volunteers.     

Novel high-performance liquid chromatographic and solid-phase extraction methods for quantitating methadone and its metabolite in spiked human urine

A novel solid-phase extraction (SPE) method and HPLC method were developed for the determination of methadone and its metabolite from spiked human urine. For sample cleanup, a spiked urine sample was pretreated with phosphoric acid followed by a well-thought-out SPE method using a 10-mg Oasis HLB 96-well extraction plate. In this SPE method, the concentration of methanol as well as the pH are optimized to preferentially isolate the analytes of interest from the sample matrix. Low elution volumes (200 μl) are achieved; this eliminates evaporation and reconstitution of the sample solution. Recoveries from human urine matrix were greater than 91% with RSD values less than 4.5%. For the HPLC analysis, the separation was obtained using a SymmetryShield RP₁₈ column with a mobile phase of 0.1% TFA–methanol (60:40, v/v). Good peak shapes were obtained without the need of addition of any competing reagent to the mobile phase. Additionally, significant signal-to-noise enrichment was achieved by diluting the final SPE eluates four-fold with water.