Validation of an electrospray ionization LC/MS/MS method for quantitative analysis of vincristine in human plasma samples

Vincristine is a natural vinca alkaloid widely used in paediatric cancer treatment. Vincristine pharmacokinetics has been already studied, but few data are available in paediatric populations. A sensitive and specific liquid chromatography–tandem mass spectrometry (LC/MS/MS) method was developed for the quantification of vincristine in plasma in order to investigate pharmacokinetics in a paediatric population. Two hundred microliters of plasma was added to vinblastine, used as internal standard. Chromatographic separation was achieved on a C8 HPLC column (Phenomenex Luna 50 mm × 2.0 mm, 3.0 μm) with a mobile phase gradient at a flow rate of 0.2 ml/min. Quantification was performed using the transition of 825.4 → 765.4 (m/z) for vincristine and 811.4 → 751.4 (m/z) for vinblastine. Chromatographic separation was achieved in 8 min. The limit of quantification was 0.25 ng/ml with a precision of 10.2% and an accuracy of 99.6%. The calibration curve was linear up to 50.0 ng/ml. Intra-day precision and accuracy ranged from 6.3% to 10% and from 91.9% to 100.8%, respectively. Inter-assay precision and accuracy ranged from 3.8% to 9.7% and from 93.5% to 100.5%, respectively. No significant matrix effect was observed for vincristine. A rapid, specific and sensitive LC/MS/MS method for quantification of vincristine in human plasma was developed and is now successfully applied for pharmacokinetic studies in paediatric patients.     

Liquid chromatography/tandem mass spectrometry method for the simultaneous determination of vardenafil and its major metabolite,N-desethylvardenafil,in human plasma: Application to a pharmacokinetic study

A rapid and sensitive LC–MS/MS method for the determination of vardenafil and its major metabolite, N-desethylvardenafil, in human plasma using sildenafil as an internal standard was developed and validated. The analytes were extracted from 0.25-mL aliquots of human plasma by liquid–liquid extraction, using 1 mL of ethyl acetate. Chromatographic separation was carried on a Luna C₁₈ column (50 mm × 2.0 mm, 3 μm) at 40 °C, with an isocratic mobile phase consisting of 10 mM ammonium acetate (pH 5.0) and acetonitrile (10:90, v/v), a flow rate of 0.2 mL/min, and a total run time of 2 min. Detection and quantification were performed using a mass spectrometer in the selected reaction-monitoring mode with positive electrospray ionization at m/z 489.1 → 151.2 for vardenafil, m/z 460.9 → 151.2 for N-desethylvardenafil, and m/z 475.3 → 100.1 for the internal standard (IS), respectively. This assay was linear over a concentration range of 0.5–200 ng/mL with a lower limit of quantification of 0.5 ng/mL for both vardenafil and N-desethylvardenafil. The coefficient of variation for the assay precision was <13.6%, and the accuracy was >93.1%. This method was successfully applied to a pharmacokinetic study after oral administration of vardenafil 20 mg tablet in Korean healthy male volunteers.     

A sensitive LC–MS/MS method for quantification of a nucleoside analog in plasma: Application to in vivo rat pharmacokinetic studies

A LC–MS/MS method was developed and validated for determination of nucleoside analog (NA) in rat plasma. The method run time was 6 min and the limit of quantification (LOQ) was estimated at 100 pg/mL. The extraction procedure consisted on plasma samples protein precipitation with an acetonitrile solution which contained the stable isotope labeled internal standard (IS). Chromatography was performed on a newly developed C₁₆ column (150 mm × 4.6 mm, 5 μm) in order to avoid the use ion pair salts. The samples were eluted at 0.8 mL/min with a gradient of mobile phase made of water and acetonitrile both acidified with 0.5% acetic acid and 0.025% trifluoroacetic acid (TFA). A tandem mass spectrometer was used as a detector for quantitative analysis. Intra-run and inter-run precision and accuracy within ±15% were achieved during a 3-run validation for quality control samples at four concentration levels in rat plasma, over a concentration ranging between 0.1 and 1000 ng/mL. The data indicate that our LC–MS/MS assay is an effective method for the pharmacokinetics study of NA in rat plasma.     

Effect of disease state on ionization during bioanalysis of MK-7009, a selective HCV NS3/NS4 protease inhibitor,in human plasma and human Tween-treated urine by high-performance liquid chromatography with tandem mass spectrometric detection

HPLC–MS/MS methods for the determination of a Hepatitis C NS3/NS4 protease inhibitor (MK-7009) in human plasma and Tween-treated urine were developed and validated over the concentration range 1–1000 ng/mL and 0.2–100 μg/mL respectively. A stable isotope labeled internal standard (ISTD), D₄-MK-7009, was employed. Analytes were chromatographed by reversed phase HPLC and quantified by an MS/MS system. Electrospray ionization in the positive mode was employed. Multiple reaction monitoring of the precursor to product ion pairs m/z 758.6 → 637.4 MK-7009 and m/z 762.5 → 637.4 ISTD was used for quantitation. Analyte and internal standard were extracted from 250 μL of plasma using an automated 96-well liquid–liquid extraction. Plasma pH adjustment prior to extraction minimized ionization suppression in plasma samples from patients with Hepatitis C. The urine method involved direct dilution in the 96-well format of 0.020 mL Tween-treated urine. These methods have supported several clinical studies. Incurred plasma sample reanalysis demonstrated adequate assay reproducibility and ruggedness.     

Validation and implementation of a liquid chromatography/tandem mass spectrometry assay to quantitate aminoflavone (NSC 686288) in human plasma

A reverse-phase liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) method was developed and validated for determination of aminoflavone (AF) in human plasma. Sample preparation involved a liquid–liquid extraction by the addition of 0.25 mL of plasma with 1.0 mL ethyl acetate containing 50 ng/mL of the internal standard zileuton. The analytes were separated on a Waters X-Terra? MS C₁₈ column using a mobile phase consisting of methanol/water containing 0.45% formic acid (70:30, v/v) 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 AF concentration range of 5–2000 ng/mL in human plasma. The lower limit of quantitation (LLOQ) was 5 ng/mL for AF in human plasma. The accuracy and within- and between-day precisions were within the generally accepted criteria for bioanalytical method (<15%). This method was successfully applied to characterize AF plasma concentration-time profile in the cancer patients in a phase I trial.     

Simultaneous determination of citalopram and its metabolite in human plasma by LC–MS/MS applied to pharmacokinetic study

A simple sensitive and robust method for simultaneous determination of citalopram and desmethylcitalopram was developed using liquid chromatography tandem mass spectrometry (LC–MS/MS). A 200 μL aliquot of plasma sample was employed and deproteinized with methanol and desipramine was used as the internal standard. After vortex mixing and centrifugation, the supernatant was diluted with water (1:1, v/v) and then directly injected to analysis. Analytes were separated by a Zorbax XDB C₁₈ column with the mobile phase composed of acetonitrile and water (30:70, v/v) with 0.25% formic acid and monitored in MRM mode using a positive electrospray source with tandem mass spectrometry detection. The total run time was 3.5 min. The dynamic range was 0.2–100 ng/mL for citalopram and 0.25–50 ng/mL for desmethylcitalopram, respectively. Compared to the best existing literatures for plasma samples, the same LOQ for CIT (0.5 ng/mL) and lower LOQ for DCIT (0.25 vs 5 ng/mL) were reached, and less sample preparation steps and runtime (3.5 vs 10 min) were taken for our method. Accuracy and precision was lower than 8% and lower than 11.5% for either target. Validation results and its application to the analysis of plasma samples after oral administration of citalopram in healthy Chinese volunteers demonstrated the method was applicable to pharmacokinetic studies.     

Simultaneous determination of 11 phytoestrogens in human serum using a 2 min liquid chromatography/tandem mass spectrometry method

A rapid 2 min liquid chromatography–tandem mass spectrometry (LC–MS/MS) method operating in multiple reaction ion monitoring mode was developed and validated that allows for the characterization and simultaneous quantification of 11 phytoestrogen metabolites with mass transitions m/z 241/119 (equol), 253/132 (daidzein), 255/149 (dihydrodaidzein), 257/108 (O-desmethylangolesin), 269/133 (genistein), 283/184 (glycitein), 267/191 (formononetin), 289/109 (biochanin A), 267/91 (coumestrol), enterodiol (301/253), and enterolactone (297/253). The method was demonstrated to be specific and sensitive, and a linear response for each phytoestrogen was observed over a range of 1–5000 ng/mL in human serum with the exception of dihydrodaidzein, whose lower limit of quantification was 2 ng/mL. The separation was carried out on a Synergi Polar-RP 2.5 micron (50 mm × 2.0 mm i.d.) column at 50 °C with water and acetonitrile (both containing 10 mM ammonium acetate) as the mobile phase under gradient conditions at a flow rate of 0.75 mL/min. This LC–MS/MS method is very useful for high-throughput analysis of phytoestrogens and proved to be simple, sensitive, reproducible, and reliable.     

Simultaneous determination of didanosine and its amino acid prodrug,valdidanosine by hydrophilic interaction chromatography coupled with electrospray ionization tandem mass spectrometry: Application to a pharmacokinetic study in rats

A rapid, sensitive and selective ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method with hydrophilic interaction chromatography has been developed and validated for the simultaneous determination of didanosine and valdidanosine (L-valine amino acid ester prodrug of didanosine) in rat plasma. Solid-phase extraction (SPE) column was employed to extract the analytes from rat plasma, with high extraction recovery (>85%) for both didanosine and valdidanosine. The analytes were then separated by hydrophilic interaction chromatography (HILIC column) and detected by a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source. The method was linear over the concentration ranges of 2–20,000 ng/mL for didanosine and 4–300 ng/mL for valdidanosine. The lower limit of quantitation (LLOQ) of didanosine and valdidanosine was 2 and 4 ng/mL, respectively. The intra-day and inter-day relative standard deviation (RSD) were less than 15% and the relative errors (RE) were all within 15%. Finally, the validated UPLC–MS/MS method was successfully applied to the pharmacokinetic study after either didanosine or valdidanosine orally administrated to the Sprague–Dawley rats.     

Characterisation of novel angiotensin-I-converting enzyme inhibitory tripeptide,Gly-Val-Arg derived from mycelium of Pleurotus pulmonarius

It has been shown that fraction D₆ from Pleurotus pulmonarius has the potential to inhibit ACE. After this discovery, additional studies were conducted to obtain peptides from that fraction, as ACE inhibitors. By size exclusion chromatography, single peak was resolved and termed as Psec. The IC₅₀ of Psec was assessed to be 4.50 μg/mL, which was 2.5 times lower than that of D₆. When Psec was resolved by SDS-PAGE, three bands with estimated molecular weight of 63 kDa, 55 kDa and 11 kDa were observed. The protein bands were subjected to MALDI-Tof MS/MS for protein identification. By using the BIOPEP database for predicting in silico digestion of gastrointestinal (GI) enzymes, four stable tripeptides with ACE inhibitor potential resulting from GI enzyme digestion were identified, namely GVR, VVR, NPR, and VVL. The IC₅₀ was estimated to be 55 μg/mL, 93 μg/mL, 110 μg/mL and >250 μg/mL individually. Based on a Lineweaver-Burk plot, tripeptide GVR was determined to be a competitive inhibitor and this was confirmed by molecular docking analysis. At 100 mg/kg of body weight (bw), the tripeptide GVR reduced SBP 33.5 mm Hg in SHRs. The results suggested that this tripeptide is potentially beneficial as an antihypertensive agent.     

Simultaneous determination of tolbutamide,omeprazole, midazolam and dextromethorphan in human plasma by LC–MS/MS—A high throughput approach to evaluate drug–drug interactions

Drug–drug interactions involving cytochrome P450 (CYP450s) are an important factor for evaluation of a new chemical entity (NCE) in drug development. To evaluate the potential inhibitory effects of a NCE on the pharmacokinetics of a cocktail of representative probes of CYP enzymes (midazolam for CYP3A4, tolbutamide for CYP2C9, omeprazole for CYP2C19 and dextromethorphan for CYP2D6) and the safety and tolerability of the NCE in the presence of probe substrates, a high throughput liquid chromatography/tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of tolbutamide, omeprazole, midazolam and dextromethorphan in human plasma using tolbutamide-d₉, midazolam-d₄, (±)-omeprazole-d₃, and dextromethorphan-d₃ as the internal standards (ISs). Human plasma samples of 50 μL were extracted by a simple protein-precipitation procedure and analyzed using a high performance liquid chromatography electrospray tandem mass spectrometer system. Reversed-phase HPLC separation was achieved with a Hypersil GOLD AQ column (50 mm × 4.6 mm, 5 μm). MS/MS detection was set at mass transitions of 271 → 172 m/z for tolbutamide, 346 → 198 m/z for omeprazole, 326 → 291 m/z for midazolam, 272 → 171 m/z for dextromethorphan, 280 → 172 m/z for tolbutamide-d₉ (IS), 349 → 198 m/z for (±)-omeprazole-d₃ (IS), 330 → 295 m/z for midazolam-d₄ (IS), and 275 → 171 m/z for dextromethorphan-d₃ (IS) in positive mode. The high throughput LC–MS/MS method was validated for accuracy, precision, sensitivity, stability, recovery, matrix effects, and calibration range. Acceptable intra-run and inter-run assay precision (<10%) and accuracy (<10%) were achieved over a linear range of 50–50,000 ng/mL for tolbutamide, 1–1000 ng/mL for omeprazole, 0.1–100 ng/mL for midazolam and 0.05–50 ng/mL for dextromethorphan in human plasma. Method robustness was demonstrated by the 100% pass rate of 24 incurred sample analysis runs and all of the 50 clinical study samples used for incurred sample reproducibility (ISR) test having met the acceptance criterion (%Diff within 20%). The overall ISR results for all compounds showed that over 95% of the samples had a %Diff of less than 10%. The method is simple, rapid and rugged, and has been applied successfully to sample analysis in support of a drug–drug interaction study.     

Liquid chromatography/electrospray tandem mass spectrometry method for the determination of cefuroxime in human plasma: Application to a pharmacokinetic study

A rapid, selective and sensitive high performance liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of cefuroxime in human plasma. Cefuroxime and the internal standard (IS), cefoxitin, were extracted from plasma samples using solid phase extraction with Oasis HLB cartridges. Chromatographic separation was performed on a LiChrospher^® 60 RP Select B column (125 mm × 4 mm i.d., 5 μm particle size) using acetonitrile:5 ± 0.2 mM ammonium acetate solution:glacial acetic acid (70:30:0.020, v/v/v) as the mobile phase at a flow rate of 0.8 mL/min. Detection of cefuroxime and cefoxitin was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in negative ion mode. The calibration curves were linear over the range of 81.0–15976.2 ng/mL with the lower limit of quantitation validated at 81.0 ng/mL. The intra- and inter-day precisions were within 7.6%, while the accuracy was within ±6.3% of nominal values. No matrix effect was observed in this method. The validated LC–MS/MS method was successfully applied for the evaluation of pharmacokinetic and bioequivalence parameters of cefuroxime after an oral administration of 500 mg cefuroxime tablet to 36 healthy male volunteers.     

Simultaneous determination of bisoprolol and hydrochlorothiazide in human plasma by HPLC coupled with tandem mass spectrometry

A sensitive, specific and selective method has been developed for the simultaneous determination of bisoprolol and hydrochlorothiazide in human plasma. The method employed a state of the art LC–MS/MS operated in the positive and negative ionization switching modes. A simple sample preparation step involving protein precipitation with acetonitrile has been optimized; the analytes and the internal standard moxifloxacin were separated on a Purosphere^® STAR C₈ column (125 mm × 4 mm, 5 μm). The mobile phase was an ammonium acetate solution (1 mM) with formic acid (0.2%): methanol and acetonitrile (65:17.5:17.5, v/v/v (%)), the flow rate was set at 0.65 mL/min. Bisoprolol and hydrochlorothiazide were ionized using ESI source prior to detection by Multiple Reaction Monitoring (MRM) mode while monitoring at the following transitions: positive m/z 326 → 116 for bisoprolol, negative m/z 296 → 269 and m/z 296 → 205 for hydrochlorothiazide. Linearity was demonstrated over the concentration range 0.10–30.0 (ng/mL) for bisoprolol and 1.00–80.00 ng/mL for hydrochlorothiazide. The limits of detection were 0.100 (ng/mL) for bisoprolol and 1.00 (ng/mL) for hydrochlorothiazide. The validated method was successfully applied to a pharmacokinetic study of 5 mg bisoprolol fumarate with 12.5 mg hydrochlorothiazide tablet in healthy volunteers.     

Development and validation of a high throughput and robust LC–MS/MS with electrospray ionization method for simultaneous quantitation of diltiazem and its two metabolites in human plasma: Application to a bioequivalence study

A high throughput and specific method using ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) was developed for the simultaneous determination of diltiazem and its two metabolite (N-desmethyldiltiazem and O-desacetyldiltiazem) in human plasma. A one-step liquid–liquid extraction (LLE) with methyl-t-butyl ether (MTBE) involved for the extraction of diltiazem (DLTZ), metabolites (DMeD and DAcD) and internal standard. Analytes were chromatographed on a ACQUITY UPLC? BEH C₁₈ column (100 mm × 2.1 mm, i.d., 1.7 μm) with isocratic elution at a flow rate of 0.2 mL/min using 10 mM ammonium acetate buffer–acetonitrile (25:75, v/v). The Quattro Premier XE LC–MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Using 300 μL plasma, the method was validated over the concentration range 0.48–639.9 ng/mL for DLTZ and 0.24–320.1 for DMeD and 0.24–320.7 ng/mL for DAcD, with a lower limit of quantification of 0.48 ng/mL for DLTZ and 0.24 ng/mL for metabolites. The intra- and inter-day precision and accuracy were within 10.0%. The recovery was 77.4%, 76.0%, 74.5% and 74.1% for DLTZ, DMeD, DAcD and Ziprasidone, respectively. Total run time was 2.0 min only.     

Determination of the quaternary ammonium compound trospium in human plasma by LC–MS/MS: Application to a pharmacokinetic study

A highly sensitive, specific and evaporation free SPE extraction, LC–MS/MS method has been developed for the estimation of trospium in human plasma using trospium-d8 as an internal standard (IS). The analyte was separated using isocratic mobile phase on reverse phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M⁺] cations, m/z 392–164 for trospium and m/z 400–172 for the IS. The total run time was 3.50 min and the elution of trospium and trospium-d8 (IS) occurred at 2.8 min. The developed method was validated in human plasma with a lower limit of quantification of 0.05 ng/mL. A linear response function was established for the range of concentrations 0.05–10 ng/mL (r > 0.998) for trospium in human plasma. The intra- and inter-day precision values for trospium met the acceptance as per FDA guidelines. Trospium was stable in the battery of stability studies viz., bench-top, auto-sampler, dry extracts and freeze/thaw cycles. The developed assay method was applied to an oral pharmacokinetic study in humans.     

Annual sex steroid and other physiological profiles of Pacific lampreys (Entosphenus tridentatus)

We documented changes in plasma levels of estradiol 17-β (E2), progesterone (P), 15α-hydroxytestosterone (15α-T), thyroxine (T4), triiodothyronine (T3), protein, triglycerides (TGs), and glucose in adult Pacific lampreys (Entosphenus tridentatus) held in the laboratory in two different years. Levels of E2 in both sexes ranged from 0.5 to 2 ng/mL from September to March, peaked in late April (2–4 ng/mL), and decreased in May, with levels higher in males than in females. Levels of P were low from September through April, but then increased substantially during May (2–4 ng/mL), with levels again highest in males. Levels of 15α-T in males were around 0.75 ng/mL through the winter before exceeding 1 ng/mL in April and decreasing thereafter, whereas females showed a gradual increase from 0.25 ng/mL in November to 0.5 ng/mL in April before decreasing. Thyroxine concentrations differed between fish in each year, with most having levels ranging from 0.75 to 2.5 ng/mL in the fall and winter, and only fish in 2003 showing distinct peaks (3–4 ng/mL) in early April or May. Plasma T3 was undetectable from November through mid-March before surging dramatically in April (ca. 150 ng/mL) and decreasing thereafter. Levels of protein, TGs, and glucose decreased or were stable during the fall and winter with TGs and glucose surging in late April to early May for some fish. Our study is the first to document long-term physiological changes in Pacific lampreys during overwintering and sexual maturation and increases our understanding of the life history of this unique fish.     

A sensitive and high-throughput LC–MS/MS method for the quantification of pegylated-interferon-α2a in human serum using monolithic C18 solid phase extraction for enrichment

The analysis of pegylated-interferon-α_2a in patient serum samples is of high interest for clinic research trials, as this therapeutic protein has become an important antiviral treatment. In this study, an LC–MS/MS method for the absolute quantification of pegylated-interferon-α_2a in human serum was developed. The assay achieved a lower limit of quantification of 3.6 ng/mL (60 pM) with the use of a monolithic C₁₈ solid phase extraction to enrich the target protein. The linear range of the assay was defined up to 54 ng/mL to measure the typical clinical pegylated-interferon-α_2a levels, and within this range, the precision and accuracy were found to be within ±20%. The method was applied to a clinical study and found suitable for high-throughput analysis of pegylated-interferon-α_2a in human serum. In addition, further investigations suggested the enrichment step may have general application to the sensitive analysis of other low molecular weight proteins.     

Liquid chromatography and tandem mass spectrometry for the quantitative determination of ixabepilone (BMS-247550, Ixempra™) in human plasma: Method validation,overcoming curve splitting issues and eliminating chromatographic interferences from degradants

A sensitive method was developed and validated for the measurement of ixabepilone (BMS-247550, Ixempra?) using a demethylated analogue of ixabepilone (BMS-212188) as an internal standard. A 0.050 mL portion of each plasma sample was extracted with 0.450 mL of acetonitrile containing the internal standard via protein precipitation. The supernatant was analyzed on a LC–MS/MS system. Chromatography was carried out on a 2.0 mm × 100 mm YMC ODS-AQ 3 μm column using an isocractic mobile phase consisting of acetonitrile:10 mM ammonium acetate, pH 5.0 (70:30, v/v) at a flow rate of 0.30 mL/min. The mass spectrometer was fitted with a TurboIonSpray^® source and operated in negative ionization mode. Detection of ixabepilone and BMS-212188 were accomplished using multiple reaction monitoring (MRM) of precursor > product ion pairs of m/z 505.2 > 405.2, and 492.1 > 392.1, respectively. The assay range was 2.00–500 ng/mL and was fitted to a 1/x² weighted quadratic regression model. Replicate sample analysis indicated that intra- and inter-day accuracy and precision are within ±15.0%. The recovery of ixabepilone from 0.050 mL of plasma containing 5.00 and 400 ng/mL was greater than 94%. The method was demonstrated to be sensitive, selective and robust, and was successfully used to support clinical studies. This paper also discussed approaches used for resolving a curve splitting issue observed during quantitative analysis of ixabepilone in biological matrices. Finally, to adapt the methodology to pharmacokinetics of ixabepilone after oral administration, the potential interference of chemical degradants on the determination of ixabepilone was evaluated.     

Validation and clinical application of a high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of 10 anti-retrovirals in human peripheral blood mononuclear cells

This paper reports the validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method that allows the quantification of 10 antiretroviral (ARV) drugs in peripheral blood mononuclear cells (PBMCs) using 6 different isotopic internal standards (IS) and its clinical application. PBMCs are isolated from blood by density gradient centrifugation and drugs are extracted with a 60% methanol (MeOH) solution containing the 6 IS. The cell extract is then injected in the HPLC system and analytes are separated on a Symmetry Shield RP18 2.1 mm × 50 mm column. The different molecules are then detected by MS/MS in electrospray positive or negative ionisation modes and data are recorded using the multiple reaction monitoring (MRM) mode. Calibration curves are constructed in the range of 0.25–125 ng/ml of cell extract by a 1/x² weighted quadratic regression. The regression coefficients obtained are always greater than 0.99 and back calculated values always comprised in the range of ±15% from their nominal concentration. Mean extraction recoveries are greater than 80% for all analytes and the method is accurate and precise with CV and bias lower than 9.4%. The lower limits of quantification (LLOQ) of the different drugs range from 0.0125 to 0.2 ng/ml of cell extract. This method was successfully applied to a cohort of 98 HIV-infected patients treated with Kaletra^® (400/100 mg of lopinavir/ritonavir (LPV/RTV) twice a day, n = 48) or with Stocrin^® (600 mg once a day, n = 50) and has been tested for cellular quantification of tipranavir (TPV) in 2 patients treated with Aptivus^® (500 mg twice a day). The patients treated by Kaletra^® showed mean cell-associated concentrations (CC) of 1819.0 and 917.2 ng/ml, for LPV and RTV, respectively. Patients treated with Stocrin^® showed mean CC of 2388.11 ng/ml while both patients under Aptivus^® showed TPV CC of 4322.7 and 1078.0 ng/ml, respectively. This method can be used to analyze ARV drug concentrations within the target tissue.     

Quantitative determination of formononetin and its metabolite in rat plasma after intravenous bolus administration by HPLC coupled with tandem mass spectrometry

A new simple, rapid, sensitive and accurate quantitative detection method using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) for the measurement of formononetin (FMN) and daidzein (DZN) levels in rat plasma is described. Analytes were separated on a Supelco Discovery C18 (4.6 × 50 mm, 5.0 μm) column with acetonitrile: methanol (50:50, v/v) and 0.1% acetic acid in the ratio of 90:10 (v/v) as a mobile phase. The method was proved to be accurate and precise at linearity range of 5–100 ng/mL with a correlation coefficient (r) of ≥0.996. The intra- and inter-day assay precision ranged from 1.66–6.82% and 1.87–6.75%, respectively; and intra- and inter-day assay accuracy was between 89.98–107.56% and 90.54–105.63%, respectively for both the analytes. The lowest quantitation limit for FMN and DZN was 5.0 ng/mL in 0.1 mL of rat plasma. Practical utility of this new LC–MS/MS method was demonstrated in a pharmacokinetic study in rats following intravenous administration of FMN.