Rapid and simple method for the determination of zolpidem in human plasma by high-performance liquid chromatography

A simple and reproducible method for the determination of zolpidem in human plasma is presented. This method involves protein precipitation with methanol (2 ml of methanol are added to 0.5 ml of plasma) and reversed-phase chromatography with fluorescence detection (excitation wavelength 244 nm, emission wavelength 388 nm). The mobile phase consists of methanol–30 mM dihydrogen potassium phosphate–triethylamine (30:69:1). pH of the aqueous part of the mobile phase is 6.8. No internal standard is required. Limit of quantitation is 1.5 ng/ml and the calibration curve is linear up to 400 ng/ml. Within-day and between-day precision expressed by relative standard deviation is less than 5% and inaccuracy also does not exceed 9%. The assay is useful for pharmacokinetic studies.     

Fluorodensitometric evaluation of gentamicin from plasma and urine by high-performance thin-layer chromatography

High-performance thin-layer chromatographic (HPTLC) analysis of gentamicin by in situ fluorodensitometric evaluation of its 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) derivative is presented. The aminoglycoside components separated on silica gel plates using chloroform–methanol–20% ammonium hydroxide (2.4:2.2:1.5, v/v/v) as the mobile phase were reacted with NB-Cl to yield highly fluorescent derivatives. The calibration curves of gentamicin in water, plasma and urine were linear in the range 40–200 ng. The mean values of intercept, slope and correlation coefficient were 16.82±0.473, 6.83±0.015 and 0.9968±0.0017 for standard curves in water, 17.35±0.375, 6.85±0.018 and 0.9941±0.0012 for standard curves in plasma and 14.35±0.286, 6.86±0.002 and 0.9933+0.0011 for standard curves in urine respectively. The analytical technique was validated for within-day and day-to-day variation. The results indicate that HPTLC, coupled with in situ fluorodensitometry, is a reliable and valuable technique for quantitative analysis of the bulk drug gentamicin and gentamicin from urine and plasma.     

Development and validation of a liquid chromatography–mass spectrometry method for the determination of verticinone in rat plasma and its application to pharmacokinetic study

We have developed and validated a sensitive liquid chromatography–electrospray ionization-mass spectrometric (LC–ESI-MS) method for the quantification of verticinone, a major active constituent from Fritillaria hupehensis Hsiao et KC Hsia., in rat plasma. Verticinone and the internal standard (IS), hupehenine, were extracted from plasma samples by a simple liquid–liquid extraction with ethyl acetate after being alkalified by 1 M ammonia hydroxide. Chromatographic separation was achieved on a C₁₈ column using a gradient elution program with methanol and water as the mobile phase. The detection was performed by selected ion monitoring (SIM) mode via positive electrospray ionization (ESI) interface. The lower limit of quantification (LLOQ) was 0.1 ng/mL. The calibration curves were linear (r² > 0.998) over the concentration range of 0.1–200 ng/mL. Within- and between-run precision was less than 6.5% and accuracy was within ±10.7%. The validated method was applied to the pharmacokinetic study of verticinone in rats after a single oral administration of 1 mg/kg.     

Determination of 2-hydroxyflutamide in human plasma by liquid chromatography–tandem mass spectrometry (LC–MS/MS): Application to a bioequivalence study on Chinese volunteers

A sensitive, simple and rapid ultra fast liquid chromatography (UFLC)–ESI-MS/MS method was developed for the determination of 2-hydroxyflutamide in human plasma using tegafur as the internal standard. The plasma sample was pretreated with methanol for protein precipitation and the analytes were separated on an Ultimate C18 column (5 μm, 2.1 mm × 50 mm, MD, USA) with the mobile phase consisted of acetonitrile and water (2:1, v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer under a negative multiple reaction-monitoring mode (MRM). The mass transition ion-pair was followed as m/z 290.90–204.8 for 2-hydroxyflutamide and 198.9–128.8 for tegafur. Linear calibration curves were obtained in the concentration range of 1.742–1452 ng/ml with a lower limit of quantification of 1.742 ng/ml. The intra- and inter-batch precision values were less than 8.1% and 5.6%, respectively. The established method was successfully applied to a bioequivalence study of two flutamide preparations (250 mg) in 20 healthy male volunteers.     

Determination of medrogestone in plasma by high-performance liquid chromatography

Interference with the UV absorbance of medrogestone by endogenous steroids in plasma was prevented by reacting plasma with oxalyl chloride. The reduction of interference was effective when oxalyl chloride was in the range 10–50 μl/ml plasma. Reaction of oxalyl chloride with plasma for 10 min could reduce interference approximately 5.5-fold, and there was no significant reduction after 30 min. The limit of quantitative concentration for medrogestone in HPLC was 1 ng/ml. The standard curves were linear with the correlation coefficient greater than 0.999 in the range of 1–30 ng/ml. The coefficients of variation of both intra- and inter-day mean values were <12% and <10% of the actual values, respectively. The developed method for plasma sample preparation and the evaluated HPLC condition were further applied to an in vivo pharmacokinetic study.     

A fast and sensitive HPLC-MS/MS analysis and preliminary pharmacokinetic characterization of cudratricusxanthone B in rats

A method for the quantitative analysis of cudratricusxanthone B (CXB) in rat plasma by high performance liquid chromatography–electrospray ionization-tandem mass spectrometry (HPLC–ESI-MS/MS) has been developed and validated. The method involved liquid–liquid extraction from plasma, simple chromatographic conditions on a Venusil XBP-PH C₁₈ column with the mobile phase of 0.5% formic acid in methanol, and mass spectrometric detection using an API-3000 instrument. Multiple reaction monitoring (MRM) mode was used to monitor precursor/product ion transitions of m/z 397.1/285.0 for CXB and m/z 381.6/269.2 for the internal standard (I.S.) cudraxanthone H. The standard curves were linear over the concentration range of 1–500 ng/mL for CXB in rat plasma. The intra- and inter-batch accuracy for CXB at four concentrations was 89.4–99.5% and 89.4–100.8%, respectively. The RSDs were less than 7.92%. The lower limit of quantification for CXB was 1.0 ng/mL using 100 μL of plasma. The average extraction recoveries of CXB ranged from 80.1 to 95.4% at the concentrations of 2, 50 and 500 ng/mL, respectively. This method was successfully applied to the pharmacokinetic study after an intravenous administration of CXB in male Sprague–Dawley (SD) rats.     

Rapid and sensitive high-performance liquid chromatographic assay for 6-hydroxychlorzoxazone and chlorzoxazone in liver microsomes

A high-performance liquid chromatographic assay was developed for the quantitation of chlorzoxazone and its major metabolite 6-hydroxychlorzoxazone. These compounds along with phenacetin, the internal standard, were extracted from incubation mixtures using ether extraction. The extracts were analyzed on a Brownlee Spheri-5 C₈ column with a mobile-phase of acetonitrile–0.5% phosphoric acid (30:70, v/v). The assay utilized UV detection at 287 nm which provided sensitivity and specificity to simultaneously quantify chlorzoxazone and 6-hydroxychlorzoxazone from liver microsomal samples at amounts of 10 ng and greater. The mean correlation coefficient of the standard curves for 6-hydroxychlorzoxazone and chlorzoxazone was 0.998 and 0.993, respectively, over the range of 25–400 ng, and the regression curves were found to be linear at least through 1600 ng. All components eluted within 7 min, resulting in a total analysis time of 8 min. The inter-day and intra-day coefficients of variation were <7 and <3%, respectively. This method provides a rapid, sensitive and cost-effective assay for 6-hydroxychlorzoxazone and chlorzoxazone in liver microsomal incubations.     

High-performance liquid chromatographic–electrochemical assay for the quantitation of BMS-181885 in monkey plasma

A high-performance liquid chromatographic–electrochemical assay was developed and validated for the quantitation of BMS-181885 (I), an anti-migraine agent, in monkey plasma. The assay involved a solid-phase extraction of I and BMY-46317 (internal standard; I.S.) on a 1-ml cyano cartridge using the automatic solid-phase extraction cartridge (ASPEC) system. Immediately following the conditioning of the cyano column (3 ml of methanol and 2 ml of 1% glacial acetic acid), plasma (0.25 ml) was loaded on to the column. The column was then washed with a 3 ml of 0.1 M ammonium acetate buffer (pH 6). The final elution of the analytes was performed using 2 ml of methanol. The eluate was then evaporated to dryness (gentle stream of nitrogen at 40°C) and the residue was dissolved in the mobile phase and injected on to a YMC basic column (15 cm×4.6 mm; 5 μm particle size) at a flow-rate of 1 ml/min. A mixture of 0.1 M ammonium acetate at pH 6–acetonitrile–methanol (70:20:10, v/v) was used as the mobile phase. Standard curves, with a lower limit of quantitation of 2 ng/ml of I were linear (r²≥0.998; range: 2–50 ng/ml). Based on the analysis of the quality control (QC) samples, the assay was both accurate and precise. The stability of I was established following freeze–thaw cycles and storage at or below −20°C. The extraction recovery of I from monkey plasma was about 82%. The validated assay method was applied to determine the pharmacokinetics of I in monkeys following a single 1 mg/kg intravenous dose.     

Hydrophilic interaction liquid chromatography-tandem mass spectrometry for the determination of adefovir in human plasma and its application to a pharmacokinetic study

A selective, rapid and sensitive hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) method was developed for the first time to determine adefovir in human plasma and applied to a pharmacokinetic study. Plasma samples were prepared by protein precipitation with methanol followed by a further cleaning using dichloromethane. The chromatographic separation was carried out on an ACQUITY UPLC™ BEH HILIC column with the mobile phase of methanol–water–formic acid (85:15:0.2, v/v/v). The detection was performed on a triple-quadrupole tandem mass spectrometer with multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The method was rapid with a run time of 3 min per sample. The linear calibration curves were obtained in the concentration range of 1.02–102 ng/mL (r² ≥ 0.99) with the lower limit of quantification (LLOQ) of 1.02 ng/mL. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were below 12% and the accuracy (relative error, R.E.) was from 0.6% to 3.2% at all quality control (QC) levels. The method was applicable to clinical pharmacokinetic study of adefovir in healthy volunteers after oral administration of adefovir dipivoxil tablet.     

Development of high performance liquid chromatography/electrospray ionization mass spectrometry for assay of ginkgolic acid (15:1) in rat plasma and its application to pharmacokinetics study

A highly sensitive HPLC–ESI-MS method has been developed and validated for the quantification of ginkgolic acid (15:1) in a small quantity of rat plasma (50 μL) using its homologous compound ginkgolic acid (17:1) as an internal standard. GA (15:1) and GA (17:1) were extracted from biological matrix by direct protein precipitation with 5-fold volume of methanol and separated on an Elite hypersil BDS C₁₈ column (2.1 × 100 mm, 3 μm), eluted with acetonitrile:water (92:8, v/v, containing 0.3% glacial acetic acid). Linear range was 8–1000 ng/mL with the square regression coefficient (r²) of 0.996. The lowest concentration (8 ng/mL) in the calibration curve was estimated as LLOQ with both deviation of accuracy and RSD of precision <20% (n = 6). The intra- and inter-day precision ranged from 3.6% to 9.9%, and the intra- and inter-day accuracy was between 89.9% and 101.3%. This method was successfully applied to study pharmacokinetics of GA (15:1) in rats after oral administration at a dose of 10 mg/kg. GA (15:1) pharmacokinetic parameters C_max, T_max, t_1/2, AUC_0–12h are 1552.9 ± 241.0 ng/mL, 0.9 ± 0.7 h, 5.5 ± 2.6 h, 3356.0 ± 795.3 ng h/mL, respectively.     

Determination of nicotine and cotinine in human plasma by liquid chromatography-tandem mass spectrometry with atmospheric-pressure chemical ionization interface

Here we report a sensitive liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method capable of quantifying nicotine down to 1 ng/ml and cotinine to 10 ng/ml from 1.0 ml of human plasma. The method was validated over linear ranges of 1.0–50.0 ng/ml for nicotine and 10.0–500.0 ng/ml for cotinine, using deuterated internal standards. Compounds were simply extracted from alkalinized human heparinized plasma with methylene chloride, reconstituted into a solution of acetonitrile, methanol and 10 mM ammonium acetate (53:32:15, v/v) after the organic phase was dried down, and analyzed on the LC-MS-MS, which is a PE Sciex API III system equipped with a Keystone BDS Hypersil C₁₈ column and atmospheric pressure chemical ionization (APCI) interface. The between-run precision and accuracy of the calibration standards were ≤6.42% relative standard deviation (R.S.D.) and ≤11.8%n relative error (R.E.) for both nicotine and cotinine. The between-run and within-run precision and accuracy of quality controls. (2.5, 15.0, 37.5 ng/ml for nicotine and 25.0, 150.0, 375.0 ng/ml for cotinine), were ≤6.34% R.S.D. and ≤7.62% R.E. for both analytes. Sample stabilities in chromatography, in processing and in biological matrix were also investigated. This method has been applied to pharmacokinetic analysis of nicotine and cotinine in human plasma.     

Determination of the unstable drug otilonium bromide in human plasma by LC–ESI-MS and its application to a pharmacokinetic study

Otilonium bromide (OB) degrades rapidly in plasma and readily undergoes hydrolysis by the plasma esterase. In this paper, an LC–ESI-MS method has been developed for the determination of OB in human plasma. The rapid degradation of OB in plasma was well prevented by immediate addition of potassium fluoride (KF, an inhibitor of plasma esterase) to the freshly collected plasma before prompt treatment with acetonitrile. The method was validated over the concentration range of 0.1–20 ng/ml. The data of intra-run and inter-run precision and accuracy were within ±15%. The mean extraction recoveries for OB and the internal standard were higher than 93.0% and the matrix effects were negligible. The method has been successfully used in a pharmacokinetic study.     

Determination of cisapride and norcisapride in human plasma using high-performance liquid chromatography with ultraviolet detection

A simple, rapid and reproducible high-performance liquid chromatographic assay for cisapride and norcisapride in human plasma is described. Samples of plasma (150 μl) were extracted using a C₁₈ solid-phase cartridge. Regenerated tubes were eluted with 1.0 ml of methanol, dried, redissolved in 150 μl of methanol and injected. Chromatography was performed at room temperature by pumping acetonitrile–methanol–0.015 M phosphate buffer pH 2.2–2.3 (680:194:126, v/v/v) at 0.8 ml/min through a C₁₈ reversed-phase column. Cisapride, norcisapride and internal standard were detected by absorbance at 276 nm and were eluted at 4.3, 5.3 and 8.1 min, respectively. Calibration plots in plasma were linear (r>0.998) from 10 to 150 ng/ml. Intraday precisions for cisapride and norcisapride were 3.3% and 5.4%, respectively. Interday precisions for cisapride and norcisapride were 9.6% and 9.0%, respectively. Drugs used which might be coadministered were tested for interference.     

High-performance liquid chromatographic method for the determination of AG-331, a novel anti-cancer agent,in human serum and urine using solid-phase extraction and photodiode-array detection

A reversed-phase isocratic high-performance liquid chromatographic method has been developed for the determination of AG-331, a novel thymidylate synthase inhibitor, in human serum and urine. The method involves a solid-phase extraction from C₁₈ cartridges without addition of an internal standard. The methanol eluent is evaporated under nitrogen at 40°C, and reconstituted in mobile phase, acetonitrile-water (35:65, v/v) containing 25 mM ammonium phosphate. Separation of AG-331 was obtained on a C₁₈ column at a flow-rate of 1 ml/min. Chromatographic signals were monitored by a photodiode-array detector at a primary wavelength of 457 nm with a bandwidth of 4.8 nm. Standard curves are linear in the range of 22–2175 ng/ml in plasma and 44–2175 ng/ml in urine, respectively. The extraction recovery ranged from 92.9–102.4%. Intra-day coefficient of variation was less than 9.5%, and inter-day coefficient of variation was less than 14.3% for an AG-331 concentration of 44 ng/ml. This method has been used to characterize the pharmacokinetics of AG-331 in cancer patients as part of ongoing Phase I trials.     

Quantitative analysis of Z-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl)phenoxy]ethanol in human plasma using high-performance liquid chromatography

A highly sensitive and precise high-performance liquid chromatography (HPLC) assay was developed and validated for the quantitation of Z-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl) phenoxy]ethanol (FC-1271a) in human plasma. Plasma samples (1.0 ml) containing FC-1271a and internal standard (toremifene citrate; Fareston^®) were extracted using a 2% 1-butanol, 98% hexane solution with an extraction efficiency of >97%. Samples were reconstituted in methanol, irradiated with high intensity ultraviolet light (254 nm) for 1 min, and injected onto a C₁₈ reverse phase column. Samples were eluted isocratically at a flow-rate of 0.5 ml/min with a mobile phase consisting of 6.5% water and 0.5% triethylamine in methanol. The fluorescence of photochemically activated compounds was detected using a fluorometer set at an excitation wavelength of 266 nm and emission wavelength of 370 nm. Under these assay conditions, standard calibration curves were linear through a concentration range of 10–400 ng/ml. In summary, we have developed and validated an HPLC assay to quantitate FC-1271a in human plasma.     

Improved high-performance liquid chromatographic analysis of teniposide in human plasma

A simple and practical high-performance liquid chromatographic analysis has been developed for measuring teniposide (VM26) in human plasma. The present analytical method has improved extraction efficiency from human plasma, therefore allowing determination of VM26 in a clinical setting using ultraviolet detection alone. Furthermore, sample preparation was simplified and shortened through use of a one-step extraction procedure. VM26 and internal standard (ibuprofen) were extracted from human plasma (0.5 ml) with ethyl acetate. A phenyl μBondapak column eluted with a mobile phase, consisting of acetonitrile–distilled water–acetic acid (30:68:2, v/v/v) was used for separation, and quantitation was achieved with a UV monitor set at 240 nm. Average extraction efficiency was 96.8±6.6% for VM26 between 1 and 25 μg/ml, and 91.4±4.3% for internal standard, with both intra- and inter-day coefficients of variation being less than 10%. The detection limit with a 100-μl injection was estimated at 0.2 μg/ml with a signal-to-noise ratio of 3 for VM26 in human plasma. The stability data of VM26 in plasma, standard and stock solutions were also obtained. The present method was found to be an alternative to the previously reported method with an electrochemical detection, and can be easily applied to routine clinical pharmacokinetic studies of VM26.     

Simple method for the analysis of tenoxicam in human plasma using high-performance liquid chromatography

A simple, rapid and cost-effective method for the determination of tenoxicam in human plasma is described, using ketorolac as the internal standard. The extraction procedure utilised 5% zinc sulphate and methanol. A nucleosil C₁₈ column and 35:65 acetonitrile-water phosphate buffered mobile phase (pH 2.8) were used, with ultraviolet detection at 355 nm. The assay was linear in the range 40 ng/ml-10 μg/ml, with recovery of extraction ranging from 87 to 102%. The intra- and inter-assay reproducibility had coefficients of variation of 3.9–7.7 and 1.6% respectively. The limit of detection for this method was 40 ng/ml.     

Simultaneous measurement of venlafaxine and its major metabolite, oxydesmethylvenlafaxine, in human plasma by high-performance liquid chromatography with coulometric detection and utilisation of solid-phase extraction

Venlafaxine, oxydesmethylvenlafaxine and an internal standard (paroxetine) were extracted from plasma by a solid-phase extraction technique. Chromatography was performed using isocratic reversed-phase high-performance liquid chromatography (HPLC) with coulometric endpoint detection. The standard curves were linear over the range 0–200 ng/ml for both venlafaxine and oxydesmethylvenlafaxine in plasma. The mean inter- and intra-assay coefficients of variation over the range of the standard curves were less than 10%. The absolute recovery averaged 74% for venlafaxine and 67% for oxydesmethylvenlafaxine. The sensitivity was 0.5 ng for both the analytes. Plasma profiles of the analytes following oral administration of venlafaxine, are presented.     

Determination of sumatriptan succinate in human plasma by high-performance liquid chromatography with coulometric detection and utilization of solid-phase extraction

Sumatriptan succinate (the analyte) and naloxone (the internal standard) were extracted from plasma with a solid-phase extraction technique. Chromatography and detection were performed by isocratic reversed-phase high-performance liquid chromatography with coulometric end-point detection. The standard curve was linear over the range 0–100 ng/ml of sumatriptan succinate in plasma. The reproducibility (as defined by the coefficient of variation, C.V.) over the range of the standard curve was 4.9–7.3%. The recovery averaged 83%. The sensitivity was 0.25 ng of sumatriptan on column (allowing a concentration of 0.5 ng/ml to be determined from a 1-ml plasma sample volume). Plasma profiles of the analyte following subcutaneous (s.c.) administration in eight normal male volunteers, are presented.     

Determination of ticagrelor and two metabolites in plasma samples by liquid chromatography and mass spectrometry

Rapid and sensitive analytical methods using liquid chromatography with tandem mass spectrometry (LC/MS/MS) were developed for the determination of ticagrelor, the first reversible oral platelet P2Y₁₂ receptor inhibitor, and its metabolites AR-C124910XX and AR-C133913XX in human plasma. Ticagrelor and its metabolites were extracted using protein precipitation with acetonitrile. Chromatographic separations were performed on reversed phase columns and detection using atmospheric pressure chemical ionization (APCI). Ticagrelor and AR-C124910XX were analyzed in the same assay, with the internal standard, d7-ZD6140, on a C18 column using negative ionization; AR-C133913XX analyzed separately on a phenyl column using positive ionization. Full validation of the methods was performed including selectivity, lower limit of quantification, accuracy, precision stability and incurred sample reproducibility and incurred sample stability. Total analytical run time was short (2 min). Calibration curves were established in the range 5–5000 ng/mL for ticagrelor, 2.5–2500 ng/mL for AR-C124910XX and 2–1000 ng/mL for AR-C133913XX. Lower limits of quantification for ticagrelor, AR-C124910XX and AR-C133913XX were determined to be 5, 2.5 and 2.0 ng/mL, respectively from 100 μL of human plasma. For ticagrelor, AR-C124910XX and AR-C133913XX, mean intra-batch accuracy was 91.9–109.0%, 86.8–109.2% and 100.5–112.0%, respectively; intra-batch precision was 4.0–8.4%, 5.2–16.9% and 3.9–12.3%, respectively. The methods were also applied to quantification of ticagrelor, AR-C124910XX and AR-C133913XX in rabbit, rat, mouse and marmoset, using 25 μL of animal plasma. A modified methodology was developed to quantify ticagrelor and AR-C124910XX in plasma from dog and cynomolgus monkey. Human incurred samples were found to generate consistent reproducibility and stability results. This method was successfully applied to determine plasma concentrations following administration of ticagrelor in human volunteers and patients, and animal safety evaluation studies. This validated methods has the advantages of being straightforward, robust and allows a fast throughput of samples.