Simultaneous Determination of Voriconazole and Posaconazole Concentrations in Human Plasma by High-Performance Liquid Chromatography

A simple, sensitive, and selective high-performance liquid chromatographic method for the simultaneous determination of voriconazole and posaconazole concentrations in human plasma was developed and validated. Quantitative recovery following liquid-liquid extraction with diethyl ether was achieved. Linearity ranged from 0.10 to 20.0 μg/ml for voriconazole and from 0.05 to 10.0 μg/ml for posaconazole. The intra- and interday coefficients of variation were less than 8.5%, and the lower limits of quantitation were <0.05 μg/ml.Based on the increasing number of immunosuppressed patients, a rising incidence of Aspergillus infections has been observed (15, 18). Voriconazole (VRC) and posaconazole (PSC), two broad-spectrum triazole derivatives, are the recommended antimycotics for either the treatment or the prophylaxis of invasive Aspergillus infections (4, 8). Both inhibit the cytochrome P450-dependent 14α-lanosterol demethylase, which is responsible for the synthesis of ergosterol, a key compound in the fungal cell membrane (19). VRC shows nonlinear pharmacokinetics in adults and is metabolized in the liver by CYP2C19, CYP3A4, and CYP2C9, resulting in a high interindividual variability of plasma levels (5). In contrast, PSC underlies no phase I metabolism but inhibits CYP3A4 (21). Currently, PSC is only available as an oral solution, and resorption depends strongly on gastric pH and nutrition. In order to manage possible drug interactions, to balance interindividual pharmacokinetic variability, and to ensure an effective exposure to VRC and PSC, therapeutic drug monitoring is recommended (16).Several methods for quantitation of VRC or PSC in human plasma by high-performance liquid chromatography (HPLC) have been reported (3, 6, 7, 9-14, 17, 20). Up to now, only one HPLC assay has been published for their simultaneous determination (1). This assay uses complex compositions of extractant and eluent, as well as high volumes of eluent; requires a long period of sample preparation; and is of poor sensitivity. Therefore, the aim of this work was to develop a rapid, sensitive, and economical HPLC method for the simultaneous determination of VRC and PSC in human plasma samples.VRC was provided by Pfizer (New York, NY) and PSC by Schering-Plough (Kenilworth, NJ). The internal standard (IS) quinoxaline and bovine serum albumin (BSA) powder were obtained from Sigma-Aldrich (Steinheim, Germany). Stock solutions of VRC (50.0 μg/ml) and PSC (25.0 μg/ml) were prepared in methanol and were diluted for the preparation of six combined working solutions (for VRC, 0.25, 0.50, 1.0, 2.0, 5.0, and 10.0 μg/ml, and for PSC, 0.125, 0.25, 5.0, 1.0, 2.5, and 5.0 μg/ml) and three combined quality control (QC) samples (for VRC, 0.50, 2.0, and 5.0 μg/ml, and for PSC, 0.50, 2.5, and 5.0 μg/ml). The IS working solution was prepared in methanol (20.0 μg/ml). Each solution was stored at −20°C. For the preparation of plasma standard samples, BSA solutions (5%, wt/vol) were spiked with VRC, PSC, and IS (0.80 μg/ml) to obtain the above-mentioned concentrations.Five-hundred-microliter aliquots of BSA standards or plasma samples were mixed with 200 μl of 0.1 M sodium hydroxide (Merck, Darmstadt, Germany) in 10-ml glass tubes, IS (20 μl) was added, and the solutions were briefly vortexed. After the tubes were capped, samples were extracted twice with 3 ml of diethyl ether (Merck, Darmstadt, Germany) for 5 min, followed by centrifugation at 5,000 × g for 5 min. The organic layers were transferred into glass tubes and evaporated to dryness (37°C) under a gentle stream of nitrogen. The residue was dissolved with 250 μl of the mobile phase.The HPLC system (Beckman-Coulter, Krefeld, Germany) consisted of a 126 solvent pump, a 168 UV-VIS photodiode array detector, a 508 autosampler, and 32 Karat software. A ReproSil-Pur Basic C₁₈ column (150 mm by 2 mm by 5 μm) (Dr. Maisch GmbH, Ammerbuch, Germany) protected by a C₁₈ guard column (4 mm by 2 mm; Phenomenex, Aschaffenburg, Germany) was used. The mobile phase consisted of 0.09 M aqueous ammonium phosphate monobasic (Riedel-de-Haën, Seelze, Germany) and acetonitrile (Merck, Darmstadt, Germany) (50%:50%, vol/vol) (pH 5.3). The flow rate was 0.2 ml/min, detection was at 260 nm, and the injected volume was 50 μl.Representative HPLC chromatograms are shown in Fig. ​Fig.1.1. Retention times for VRC, PSC, and IS were approximately 4.90, 14.50, and 7.50 min. No interfering endogenous peaks were detectable in the blank sample.Open in a separate windowFIG. 1.Representative HPLC chromatograms (at 260 nm) of VRC, PSC, and IS in plasma samples. (A) Drug-free 5% BSA sample (blank). (B) Five-percent BSA sample spiked with VRC at 2.00 μg/ml, PSC at 2.50 μg/ml, and IS at 0.80 μg/ml. (C) Plasma sample obtained from a patient, with VRC concentration of 1.40 μg/ml. (D) Plasma sample obtained from a patient, with PSC concentration of 2.17 μg/ml.Linearity was evaluated over a concentration range of 0.10 to 20.0 μg/ml for VRC and 0.05 to 10.0 μg/ml for PSC. Using the ratios of observed peak heights for each analyte and IS, the calibration curves showed a correlation coefficient (r²) of 0.999 for all compounds. The limit of detection, defined as the lowest detectable concentration level resulting in a signal-to-noise ratio of three (2), was determined at 12.5 and 6.25 ng/ml for VRC and PSC, respectively. The lower limit of quantitation was established for both analytes at concentrations below 0.05 μg/ml, and the upper limits of quantitation were arbitrarily set at 50.0 and 25.0 μg/ml.The intraday accuracy and precision of the method were determined by measuring nine replicates of each QC concentration on the same day. For interday accuracy and precision, the procedure was repeated on seven days. The intraday precision values of VRC and PSC were 2.22 to 4.24% and 2.11 to 8.46%, respectively. The interday precision values of the corresponding compounds were below 4.0% and 5.9% (Table ​(Table1).1). The levels of recovery of analytes and IS were estimated by comparing the peak heights of extracted QC samples with those of unextracted standard solutions. The highest recovery values achieved were 94.4% for VRC, 101.3% for PSC, and 100.3% for IS.

TABLE 1.

Intra- and interday accuracy and precision for the determination of voriconazole (VRC) and posaconazole (PSC) concentrations in spiked 5% BSA samples using HPLC

Multiplex Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Quantification in Human Plasma of Fluconazole,Itraconazole, Hydroxyitraconazole,Posaconazole, Voriconazole,Voriconazole-N-Oxide,Anidulafungin, and Caspofungin

Therapeutic drug monitoring (TDM) may contribute to optimizing the efficacy and safety of antifungal therapy because of the large variability in drug pharmacokinetics. Rapid, sensitive, and selective laboratory methods are needed for efficient TDM. Quantification of several antifungals in a single analytical run may best fulfill these requirements. We therefore developed a multiplex ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method requiring 100 μl of plasma for simultaneous quantification within 7 min of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-N-oxide, caspofungin, and anidulafungin. Protein precipitation with acetonitrile was used in a single extraction procedure for eight analytes. After reverse-phase chromatographic separation, antifungals were quantified by electrospray ionization-triple-quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. Deuterated isotopic compounds of azole antifungals were used as internal standards. The method was validated based on FDA recommendations, including assessment of extraction yields, matrix effect variability (<9.2%), and analytical recovery (80.1 to 107%). The method is sensitive (lower limits of azole quantification, 0.01 to 0.1 μg/ml; those of echinocandin quantification, 0.06 to 0.1 μg/ml), accurate (intra- and interassay biases of −9.9 to +5% and −4.0 to +8.8%, respectively), and precise (intra- and interassay coefficients of variation of 1.2 to 11.1% and 1.2 to 8.9%, respectively) over clinical concentration ranges (upper limits of quantification, 5 to 50 μg/ml). Thus, we developed a simple, rapid, and robust multiplex UPLC-MS/MS assay for simultaneous quantification of plasma concentrations of six antifungals and two metabolites. This offers, by optimized and cost-effective lab resource utilization, an efficient tool for daily routine TDM aimed at maximizing the real-time efficacy and safety of different recommended single-drug antifungal regimens and combination salvage therapies, as well as a tool for clinical research.Differences in oral drug bioavailability, altered volumes of distribution, drug-drug interactions, impaired hepatic and/or renal drug clearance, and the genetic background of hepatic metabolism contribute to the large intra- and interindividual variability of pharmacokinetics of antifungal agents in patients with life-threatening fungal infections. A given drug dosing regimen can yield very different plasma concentrations: subtherapeutic drug exposure may result in a lack of response to therapy and emergence of fungal resistance, while overexposure may increase the risk of toxicity (21, 48, 57, 59). There is therefore increasing clinical evidence of the usefulness of therapeutic drug monitoring (TDM), which allows real-time adjustment of antifungal dosing aimed at optimizing the individual drug''s pharmacokinetic/pharmacodynamic profile. This may be especially helpful for severely ill patients with multiple organ dysfunctions and comedications, when drug absorption is uncertain, in infections not responding to therapy, or when severe toxicity is suspected (1, 11, 24, 35, 46, 50, 55). Tentative recommendations for timing of measurement and therapeutic concentration intervals in TDM for azole antifungals have been proposed (1). For echinocandins, experimental in vitro and in vivo studies have determined the pharmacokinetic/pharmacodynamic parameters associated with therapeutic success. Clinical studies are ongoing for the assessment of the relationships between exposure to echinocandins and efficacy or safety. Moreover, combinations of these compounds with azoles or polyenes are used for salvage therapy of refractory infections and are being investigated for first-line therapy (13, 18, 31).Analytical methods using liquid chromatography coupled with triple-quadrupole mass spectrometry (LC-MS/MS) have been reported for the quantification of single antifungals in biological fluids, including fluconazole (FLC) (14, 34, 49), itraconazole (ITZ) (39, 62) and its active metabolite (hydroxyitraconazole [OH-ITZ]) (4, 6, 27, 53), voriconazole (VRC) (2, 15, 26, 54, 63), posaconazole (PSC) (10, 40a, 44, 52), caspofungin (CASPO) (7, 16, 40), and anidulafungin (ANI) (13). LC-MS/MS assays were recently reported for the simultaneous quantification of posaconazole, voriconazole, isavuconazole, caspofungin, anidulafungin, and micafungin in different peripheral blood compartments (19) and of itraconazole, voriconazole, and posaconazole in blood (3).Ultra-performance liquid chromatography coupled with tandem mass spectrometry (ULPC-MS/MS) allows the selective and sensitive quantification of structurally unrelated drugs in a single analytical run, resulting in substantial reductions in analytical time, turnaround time, and costs. An analytical method using a simple extraction procedure followed by simultaneous quantification of multiple antifungal agents would be most efficient for rapidly providing TDM results, including those for patients receiving combination antifungal therapy (13, 18, 31), and for maximizing lab resource utilization.We aimed at developing and validating a simple, sensitive, and robust multiplex UPLC-MS/MS method for the simultaneous analysis in human plasma of six recommended and frequently used antifungal drugs and two metabolites: fluconazole, itraconazole and its active metabolite (hydroxyitraconazole), posaconazole, voriconazole, voriconazole-N-oxide (VRC-NO; the principal metabolite of voriconazole, produced by cytochrome P450 2C19 [CYP2C19] [23, 41]), caspofungin, and anidulafungin.     

A Simple High-Performance Liquid Chromatography Method for Simultaneous Determination of Three Triazole Antifungals in Human Plasma

A rapid and simple high-performance liquid chromatography (HPLC) assay was developed for the simultaneous determination of three triazole antifungals (voriconazole, posaconazole, and itraconazole and the metabolite of itraconazole, hydroxyitraconazole) in human plasma. Sample preparation involved a simple one-step protein precipitation with 1.0 M perchloric acid and methanol. After centrifugation, the supernatant was injected directly into the HPLC system. Voriconazole, posaconazole, itraconazole, its metabolite hydroxyitraconazole, and the internal standard naproxen were resolved on a C₆-phenyl column using gradient elution of 0.01 M phosphate buffer, pH 3.5, and acetonitrile and detected with UV detection at 262 nm. Standard curves were linear over the concentration range of 0.05 to 10 mg/liter (r² > 0.99). Bias was <8.0% from 0.05 to 10 mg/liter, intra- and interday coefficients of variation (imprecision) were <10%, and the limit of quantification was 0.05 mg/liter.     

Quantification and Validation of Ertapenem Using a Liquid Chromatography-Tandem Mass Spectrometry Method

Ertapenem, a carbapenem, relies on time-dependent killing. Therapeutic drug monitoring (TDM) should be considered, when ertapenem is used in specific populations, to achieve optimal bactericidal activity and optimize drug-dosing regimens. No validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been reported using deuterated ertapenem as the internal standard. A new simple and robust LC-MS/MS method using a quadrupole mass spectrometer was developed for analysis of ertapenem in human plasma, using deuterated ertapenem as the internal standard. The calibration curve was linear over a range of 0.1 (lower limit of quantification [LLOQ]) to 125 mg/liter. The calculated accuracy ranged from −2.4% to 10.3%. Within-run coefficients of variation (CV) ranged from 2.7% to 11.8%, and between-run CV ranged from 0% to 8.4%. Freeze-thaw stability had a bias of −3.3% and 0.1%. Storage of QC samples for 96 h at 4°C had a bias of −4.3 to 5.6%, storage at room temperature for 24 h had a bias of −10.7% to −14.8%, and storage in the autosampler had a bias between −2.9% and −10.0%. A simple LC-MS/MS method to quantify ertapenem in human plasma using deuterated ertapenem as the internal standard has been validated. This method can be used in pharmacokinetic studies and in clinical studies by performing TDM.     

同位素稀释超高效液相色谱串联质谱法检测血浆18-羟皮质酮方法的建立及初步临床应用

目的 建立一种同位素稀释超高效液相色谱串联质谱法(isotope dilution ultra performance liquid chromatography tandem mass spectrometry,ID-UPLC-MS/MS)检测血浆18-羟皮质酮(18-Hydroxycorticosterone,18...     

A Simple High-Performance Liquid Chromatography Method for the Quantitation of Tricyclic Antidepressant Drugs in Human Plasma or Serum

Objective: A simple reversed-phase high performance liquid chromatography method with a variable wavelength detection has been developed for determining the commonly used tricyclic antidepressant drugs in plasma. Methods: The assay procedure involves a liquid-liquid extraction with an initial extraction into hexane:ethyl acetate after basification of the homogenate. The column used was a Supelco Hypersil 5 μm, 150 × 3.2 mm. The mobile phase was acetonitrile, methanol, and phosphate buffer (0.01M, pH 7.4) at 12:3:5 ratio (v/v/v). Results: The recoveries ranged from 89 to 108% and the day-to-day precision was 1.1 to 13% CV depending on the compound. The method is sensitive to 15 ng/mL and linear to 400 ng/mL with a 25 μL injection. Conclusion: This method is simple, sensitive, precise, inexpensive, and is currently used in our laboratory for therapeutic monitoring of tricyclics. The data generated by this method were within the range outlined by the College of American Pathologists.     

In Vitro Susceptibility of Aspergillus fumigatus to Isavuconazole: Correlation with Itraconazole,Voriconazole, and Posaconazole

Triazoles are first-line agents for treating aspergillosis. The prevalence of azole resistance in Aspergillus fumigatus is increasing, and cross-resistance is a growing concern. In this study, the susceptibilities of 40 A. fumigatus clinical isolates were tested by using the CLSI method with amphotericin B, itraconazole, voriconazole, posaconazole, and the new triazole isavuconazole. Isavuconazole MICs were higher in strains with reduced susceptibilities to other triazoles, mirroring changes in voriconazole susceptibility. Isavuconazole MICs differed depending on the Cyp51A substitution.     

Network Meta-analysis and Pharmacoeconomic Evaluation of Fluconazole,Itraconazole, Posaconazole,and Voriconazole in Invasive Fungal Infection Prophylaxis

Invasive fungal infections (IFIs) are associated with high mortality rates and large economic burdens. Triazole prophylaxis is used for at-risk patients with hematological malignancies or stem cell transplants. We evaluated both the efficacy and the cost-effectiveness of triazole prophylaxis. A network meta-analysis (NMA) of randomized controlled trials (RCTs) evaluating fluconazole, itraconazole capsule and solution, posaconazole, and voriconazole was conducted. The outcomes of interest included the incidences of IFIs and deaths. This was coupled with a cost-effectiveness analysis from patient perspective over a lifetime horizon. Probabilities of transitions between health states were derived from the NMA. Resource use and costs were obtained from the Singapore health care institution. Data on 5,505 participants in 21 RCTs were included. Other than itraconazole capsule, all triazole antifungals were effective in reducing IFIs. Posaconazole was better than fluconazole (odds ratio [OR], 0.35 [95% confidence interval [CI], 0.16 to 0.73]) and itraconazole capsule (OR, 0.25 [95% CI, 0.06 to 0.97]), but not voriconazole (OR, 1.31 [95% CI, 0.43 to 4.01]), in preventing IFIs. Posaconazole significantly reduced all-cause deaths, compared to placebo, fluconazole, and itraconazole solution (OR, 0.49 to 0.54 [95% CI, 0.28 to 0.88]). The incremental cost-effectiveness ratio for itraconazole solution was lower than that for posaconazole (Singapore dollars [SGD] 12,546 versus SGD 26,817 per IFI avoided and SGD 5,844 versus SGD 12,423 per LY saved) for transplant patients. For leukemia patients, itraconazole solution was the dominant strategy. Voriconazole was dominated by posaconazole. All triazole antifungals except itraconazole capsule were effective in preventing IFIs. Posaconazole was more efficacious in reducing IFIs and all-cause deaths than were fluconazole and itraconazole. Both itraconazole solution and posaconazole were cost-effective in the Singapore health care setting.     

Therapeutic Drug Monitoring of Posaconazole in Hematology Patients: Experience with a New High-Performance Liquid Chromatography-Based Method

Parallel administration of the proton pump inhibitor (PPI) esomeprazole has been shown to decrease oral bioavailability of posaconazole in healthy volunteers. We prospectively analyzed serum samples (n = 59) obtained from hematology patients (n = 27) under posaconazole prophylaxis. Patients treated concomitantly with pantoprazole had significantly lower posaconazole levels than patients without PPI treatment (median levels of 630 μg/liter versus 1,125 μg/liter, respectively). These results suggest that drug monitoring is relevant when posaconazole and pantoprazole are administered concomitantly.Posaconazole is a broad-spectrum antifungal triazole approved for prophylaxis and treatment of invasive mycoses in hematology patients (11, 12, 23). The drug is available as an oral suspension, and serum level measurements have shown relatively high intra- and interindividual differences in both healthy volunteers (HV) and patient populations (2, 6, 18, 25). The absorption of posaconazole can be increased 2.6-fold by administering it along with food or oral nutritional supplements (3, 13, 22). Gastric pH has also been shown to influence the bioavailability of posaconazole. For instance, parallel administration of posaconazole with cimetidine and esomeprazole has been demonstrated to decrease posaconazole exposure in HV by 40% and 32%, respectively (4, 15). However, similar effects have not been assessed in hematology patients, which would be of great importance to optimize posaconazole administration in these patients. Oral bioavailability of posaconazole has been demonstrated to be especially low in patients after hematopoietic stem cell transplantation (SCT) (10). Possible reasons are high incidences of mucositis and graft-versus-host disease and drug interactions (14, 16, 26). Data in correlation between posaconazole exposure and efficacy are limited, and prospective studies to define target concentrations are missing. Two clinical trials demonstrated that elevated posaconazole levels were associated with improved responses (26, 28). One of these trials demonstrated that mean posaconazole levels in patients who did or did not develop breakthrough infections were 611 μg/liter or 922 μg/liter, respectively (26). Based on these results, the FDA briefing document recommends target posaconazole concentrations of >700 μg/liter (7).Therapeutic drug monitoring (TDM) of posaconazole was carried out with a previously published method based on high-performance liquid chromatography (19). The method''s lower limit of quantification and limit of detection were 0.1 mg/liter and 0.05 mg/liter, respectively. A linear calibration curve from 0.1 to 5 mg/liter was obtained using a 50-μl sample. Relative standard deviations of intraday variations ranged from 2.3% to 9.4%, and intraday accuracy ranged from 88.8% to 114.8%.We prospectively analyzed serum posaconazole levels in samples obtained from consecutive inpatients under posaconazole prophylaxis at our department between November 2008 and July 2009. Samples were drawn before administration of posaconazole as morning trough values. At the time of analysis, all patients had received posaconazole for at least 5 days at a dosage of 200 mg three times daily. Serum posaconazole levels were monitored on a weekly basis. When more than one serum sample obtained from a patient was analyzed, mean posaconazole levels were used for statistics. Of those patients with parallel administration of pantoprazole, four did not receive the proton pump inhibitor (PPI) during the entire period of posaconazole prophylaxis. Depending on whether more serum samples were obtained with or without concomitant administration of pantoprazole, these patient samples were classified within the cohort with the majority of samples.Low posaconazole concentrations were defined as <700 μg/liter (7). Hepatic function impairment was defined as increased alanine transaminase (ALT) levels to higher common toxicity criteria (CTC) grades, according to the National Cancer Institute (NCI) CTC guidelines. Breakthrough infections were defined as the occurrence of proven, probable, or possible fungal infections, according to the criteria of the EORTC/MSG (5). Summary statistics were used to describe patient characteristics and measured posaconazole levels. Box plots were employed to display differences between the analyzed cohorts. Statistical significance was tested with the nonparametric Wilcoxon rank sum test. A P value of <0.05 was considered statistically significant. Statistical analyses were performed by using the R software package (21). The study was approved by the ethics committee of the University of Freiburg.Serum posaconazole levels in 27 hematology patients were analyzed (characteristics summarized in Table ​Table1).1). Fifty-nine measurements were performed, with a median of two samples per patient. Serum posaconazole levels ranged from 130 to 2,300 μg/liter (median, 740 μg/liter) (Fig. ​(Fig.11 A). Two serum levels were below the method''s limit of quantification (100 μg/liter) and were treated as 0 μg/liter for the statistical analysis. Posaconazole levels of <700 μg/liter were observed in 13/27 patients (48%). The effects of age, gender, underlying disease, and number of concomitant drugs on serum posaconazole levels were not significant (Fig. 1B to E).Open in a separate windowFIG. 1.Measured posaconazole levels in all patients (n = 27) (A), patients younger (n = 13) or older (n = 14) than 58 years (B), female (n = 15) versus male (n = 12) patients (C), patients with acute leukemia (n = 22) versus other malignant diseases (n = 5) (D), and patients with eight or less (n = 13) versus more than eight (n = 14) concomitantly applied drugs (co-drugs) (E). The boxes include the interval between the 25% and 75% quantiles, and the extremes represent the 5% and 95% quantiles. Measured values outside the latter range are displayed as small circles, and the median is shown as a horizontal line in the middle of the box.

TABLE 1.

Patient characteristics

Quantification of Modified Tyrosines in Healthy and Diabetic Human Urine using Liquid Chromatography/Tandem Mass Spectrometry

The quantification of urinary oxidized tyrosines, dityrosine (DiY), nitrotyrosine (NY), bromotyrosine (BrY), and dibromotyrosine (DiBrY), was accomplished by quadruple liquid chromatography-tandem mass spectrometry (LC/MS/MS). The sample was partially purified by solid phase extraction, and was then applied to the LC/MS/MS using multiple-reaction monitoring (MRM) methods. The analysis for the DiY quantification was done first. The residual samples were further butylated with n-butanol/HCl, and the other modified tyrosines were then quantified with isotopic dilution methods. MRM peaks of the modified tyrosines (DiY, NY, BrY, and DiBrY) from human urine were measured and the elution times coincided with the authentic and isotopic standards. The amounts of modified tyrosines in healthy human urine (n = 23) were 8.8 ± 0.6 (DiY), 1.4 ± 0.4 (NY), 3.8 ± 0.3 (BrY), and 0.7 ± 0.1 (DiBrY) µmol/mol of creatinine, respectively. A comparison of the modified tyrosines with urinary 8-oxo-deoxyguanosine, pentosidine, and N^ε-(hexanoyl)lysine was also performed. Almost all products, except for NY, showed good correlations with each other. The amounts of the modified tyrosines (NY, BrY, and DiBrY) in the diabetic urine were higher than those in the urine from healthy people.     

Cryptococcus neoformans-Cryptococcus gattii Species Complex: an International Study of Wild-Type Susceptibility Endpoint Distributions and Epidemiological Cutoff Values for Fluconazole,Itraconazole, Posaconazole,and Voriconazole

Epidemiological cutoff values (ECVs) for the Cryptococcus neoformans-Cryptococcus gattii species complex versus fluconazole, itraconazole, posaconazole, and voriconazole are not available. We established ECVs for these species and agents based on wild-type (WT) MIC distributions. A total of 2,985 to 5,733 CLSI MICs for C. neoformans (including isolates of molecular type VNI [MICs for 759 to 1,137 isolates] and VNII, VNIII, and VNIV [MICs for 24 to 57 isolates]) and 705 to 975 MICs for C. gattii (including 42 to 260 for VGI, VGII, VGIII, and VGIV isolates) were gathered in 15 to 24 laboratories (Europe, United States, Argentina, Australia, Brazil, Canada, Cuba, India, Mexico, and South Africa) and were aggregated for analysis. Additionally, 220 to 359 MICs measured using CLSI yeast nitrogen base (YNB) medium instead of CLSI RPMI medium for C. neoformans were evaluated. CLSI RPMI medium ECVs for distributions originating from at least three laboratories, which included ≥95% of the modeled WT population, were as follows: fluconazole, 8 μg/ml (VNI, C. gattii nontyped, VGI, VGIIa, and VGIII), 16 μg/ml (C. neoformans nontyped, VNIII, and VGIV), and 32 μg/ml (VGII); itraconazole, 0.25 μg/ml (VNI), 0.5 μg/ml (C. neoformans and C. gattii nontyped and VGI to VGIII), and 1 μg/ml (VGIV); posaconazole, 0.25 μg/ml (C. neoformans nontyped and VNI) and 0.5 μg/ml (C. gattii nontyped and VGI); and voriconazole, 0.12 μg/ml (VNIV), 0.25 μg/ml (C. neoformans and C. gattii nontyped, VNI, VNIII, VGII, and VGIIa,), and 0.5 μg/ml (VGI). The number of laboratories contributing data for other molecular types was too low to ascertain that the differences were due to factors other than assay variation. In the absence of clinical breakpoints, our ECVs may aid in the detection of isolates with acquired resistance mechanisms and should be listed in the revised CLSI M27-A3 and CLSI M27-S3 documents.     

Defluorinated Sparfloxacin as a New Photoproduct Identified by Liquid Chromatography Coupled with UV Detection and Tandem Mass Spectrometry

Photodegradation of sparfloxacin was observed by means of high-pressure liquid chromatography with UV detection and liquid chromatography coupled with UV detection and tandem mass spectrometry (LC-MS/MS). Three products were detected. Comparison with an independently synthesized derivative of sparfloxacin revealed the structure of one product which is believed to be 8-desfluorosparfloxacin. The second product is likely to be formed by the splitting off of a fluorine and a cyclopropyl ring. Thus, photodefluorination of quinolone antibacterial agents is found and proved for the first time by LC-MS/MS.     

Validation and Application of a Liquid Chromatography-Tandem Mass Spectrometry Method To Determine the Concentrations of Sofosbuvir Anabolites in Cells

Sofosbuvir (SOF) is a highly efficacious and well-tolerated uridine nucleotide analog that inhibits the hepatitis C virus (HCV) NS5B polymerase enzyme. SOF is administered as a prodrug, which undergoes intracellular phosphorylation by host enzymes to a monophosphate, diphosphate, and finally a pharmacologically active triphosphate. In order to fully understand the clinical pharmacology of SOF, there is a great need to determine the intracellular phosphate concentrations of the drug. We describe the validation and utilization of a method to characterize SOF''s disposition into various in vivo cell types, including hepatocytes, peripheral blood mononuclear cells (PBMC), and red blood cells (RBC). Standard bioanalytical validation criteria were applied to lysed cellular matrices, with a validated linear range of 50 to 50,000 fmol/sample for each phosphate moiety. The assay was utilized to collect the first data demonstrating concentrations of phosphorylated anabolites formed in PBMC, hepatocytes, and RBC in vivo during SOF therapy. Median concentrations in PBMC were 220 (range, 51.5 to 846), 70.2 (range, 25.8 to 275), and 859 (range, 54.5 to 6,756) fmol/10⁶ cells in the monophosphate, diphosphate, and triphosphate fractions, respectively. In contrast, RBC triphosphate concentrations were much lower than those of PBMC, as the median concentration was 2.91 (range, 1.14 to 10.4) fmol/10⁶ cells. The PBMC triphosphate half-life was estimated at 26 h using noncompartmental approaches, while nonlinear mixed-effect modeling was used to estimate a 69 h half-life for this moiety in RBC. The validated method and the data it generates provide novel insight into the cellular disposition of SOF and its phosphorylated anabolites in vivo.     

液相色谱-串联质谱法测定人血浆中二甲双胍的浓度

目的采用液相色谱-串联质谱法测定人血浆中二甲双胍的浓度。方法血浆样品用乙腈(含0.1%甲酸)沉淀蛋白后用二氯甲烷反洗后进行分析。使用Agilent C8(75 mm×4.6 mm,3.5μm)色谱柱。流动相:A泵:5 mmol/L醋酸铵(三乙胺调pH值至7.5),B泵:乙腈。线性梯度洗脱,流速0.4 mL/min。采用电喷雾离子源,多反应离子监测。用于定量分析的离子对二甲双胍为130.2/71.1,内标吗啉胍为172.2/60.2。结果线性范围为50～2 000 ng/mL,最低定量限为50 ng/mL,预处理回收率为81.7%～98.0%,二甲双胍的基质效应<9.97%,日内和日间相对标准偏差均<5.2%。结论液相色谱-串联质谱法快速、简便、灵敏度高,是一种适用于人血浆中药物浓度的测定及药物动力学和生物利用度研究的方法。     

Validation of Denaturing High Performance Liquid Chromatography as a Rapid Detection Method for the Identification of Human INK4A Gene Mutations

The incidence of melanoma is increasing rapidly in western countries. Genetic predisposition in familial and in some sporadic melanomas has been associated with the presence of INK4A gene mutations. To better define the risk for developing sporadic melanoma based on genetic and environmental interactions, large groups of cases need to be studied. Mutational analysis of genes lacking hot spots for sequence variations is time consuming and expensive. In this study we present the application of denaturing high performance liquid chromatography (DHPLC) for screening of mutations. Exons 1α, 2, and 3 were amplified from 129 samples and 13 known mutants, yielding 347 products that were examined at different temperatures. Forty-two of these amplicons showed a distinct non-wild-type profile on the chromatogram. Independent sequencing analysis confirmed 16 different nucleotide variations in Leu³²Pro; Ile49Thr; 88 del G; Gln50Arg; Arg24Pro; Met53Ile; Met53Thr; Arg58stop; Pro81Leu; Asp84Ala; Arg80stop; Gly101Trp; Val106Val; Ala148Thr; and in positions (−2) in intron 1 (C → T); and in the 3′ UTR, nucleotide 500 (C → G). No false negatives or false positives were obtained by DHPLC in samples with mutations or polymorphisms. We conclude that the DHPLC is a fast, sensitive, cost-efficient, and reliable method for the scanning of INK4A somatic or germline mutations and polymorphisms of large number of samples.     

Rapid and Sensitive Liquid Chromatography/Mass Spectrometry Assay for Caspofungin in Human Aqueous Humor

A rapid, precise, and sensitive liquid chromatography/mass spectrometry (LC/MS) method to quantify the caspofungin concentration in human aqueous humor was developed and validated. Sample preparation involved simple dilution of aqueous humor samples with acetonitrile. Azithromycin was the internal standard. Good linearity over 10 to 5,000 ng/ml was observed. The lower limit of quantification was 10 ng/ml. The intra- and interday accuracies (percent bias) were within 11%, while the intra- and interday precisions were within 6%.Current topical treatment for fungal keratitis is inadequate (13) given the lack of favorable outcomes with existing antifungal eye drops (i.e., fluconazole, amphotericin B, and natamycin) (7, 14). Accordingly, it is important to investigate the topical application of newer antifungal agents that are available only as injections. In rabbit models (5, 8), caspofungin eye drops were effective in inhibiting the progression of fungal keratitis. It remains unknown if caspofungin eye drops are able to penetrate the human eye. Studies in this area are impeded by the absence of a simple and sensitive analytical assay to quantify caspofungin in human aqueous humor.Currently, high-performance liquid chromatography (HPLC) with fluorescence or amperometric detection (4, 11, 12, 15) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) (1-3, 9, 16) are used to quantify caspofungin in biological samples. The majority of these methods, however, involve liquid-liquid extraction and require large sample volumes. Because aqueous humor samples usually have small volumes (<150 μl), these methodologies are not suitable. This study aimed to develop a rapid and sensitive LC/MS assay for caspofungin that involves simple preparation and low sample volumes.Caspofungin diacetate was provided by Merck Sharp & Dohme. Azithromycin dihydrate (internal standard [IS]) was purchased from Kopran Ltd. (Maharashtra, India). Calibration standard and quality control (QC) stock solutions of caspofungin (1 mg/ml) were prepared separately in water and stored at −80°C. IS stock solution (1 mg/ml) was freshly prepared in acetonitrile for each analysis. Working solutions of caspofungin and IS were freshly prepared by diluting the stock solutions with water and acetonitrile, respectively.The calibration curve was constructed using water due to the limited availability of blank aqueous humor (6, 10). Calibration standards were prepared by adding 30 μl IS working solution to 30 μl caspofungin working solutions. The final concentrations of calibration standards were 10, 50, 100, 500, 1,000, 2,000, and 5,000 ng/ml with the IS (500 ng/ml). All QC samples (30, 300, and 4,000 ng/ml) were freshly prepared. The blank aqueous humors were pooled to prepare aqueous humor-based low (30 ng/ml; n = 3) and high (4,000 ng/ml; n = 3) QC samples (30 μl each) to validate the water-based calibration curve.The Shimadzu LC system (Shimadzu, Japan) comprised a high-pressure gradient unit (LC-20AD pump and LC-20ADsp pump), DGU-20A₃ degasser, and CTO-10A column oven with a FCV-12AH switching valve. A Synergi Hydro-RP C₁₈ column (80 Å, 50 by 2 mm, 4 μm; Phenomenex) and guard column (4 by 2 mm, 4 μm; Phenomenex) were used. The column temperature was 30°C, and the flow rate was 0.5 ml/min. The mobile phase was Milli-Q water-formic acid (A; 100:0.1 [vol/vol]) and methanol-formic acid (B; 100:0.1 [vol/vol]). The gradient elution program was as follows: 0.0 to 2.8 min from 20% B to 30% B, 2.8 to 3.0 min from 30% B to 95% B, 3.0 to 5.0 min at 95% B, 5.0 to 5.5 min from 95% B to 20% B, and 5.5 to 8.0 min at 20% B. The mobile phase was directed to an MS system between 3.0 and 6.0 min. The run time was 8.0 min. The injection volume was 10 μl.An MS-2010EV single-quadrupole mass analyzer coupled with an electrospray ionization interface (Shimadzu, Japan) was used. The tuning voltages were fixed for the interface, the curved desolvation line (CDL), and Q-array. The detector voltage was 1.5 kV. Flow rates were 1.5 liters/min for nitrogen gas and 10 liters/min for both the nebulizer and the drying gas. The temperatures for CDL and heat block were 200°C. Data were acquired and processed using the LCMSsolutions software program (version 3.4). The protonated ions of caspofungin (m/z = 547.5 [M+2H]²⁺) and IS (m/z = 749.4 [M+H]⁺) were monitored.Caspofungin eluted at 4.68 ± 0.01 min, and the IS eluted at 4.48 ± 0.01 min (Fig. ​(Fig.1).1). Blank aqueous humor samples (n = 6) showed no interfering peaks with endogenous products or coadministered drugs used in eye surgery (phenylephrine, cyclopentolate, tropicamide, and oxybuprocaine).Open in a separate windowFIG. 1.Representative chromatogram of blank aqueous humor sample (A) or two aqueous humor samples containing a caspofungin concentration of 95.1 ng/ml or 63.8 ng/ml (with IS at 500 ng/ml) (B1 and B2, respectively) from two consenting patients who were administered one drop of 0.5% caspofungin eye drop hourly for 4 h prior to the surgery. The aqueous humor samples were removed from the participants 1.67 h and 1.17 h after the last caspofungin eye drop was administered for B1 and B2, respectively.Calibration curves plotted the peak area ratio of caspofungin to IS (y) against the caspofungin concentration (x), with y = 0.005x − 0.074 (n = 5; r² = 0.999 ± 0.003). Good linearity over 10 to 5,000 ng/ml was observed, with 1/x weighting employed. The limit of detection (LOD) calculated using the equation LOD = 3.3σ/S was 2.11 ng/ml (σ is the standard deviation of the calibration curve, and S is the slope of the calibration curve). The lower limit of quantification (LLOQ), defined as the reproducible peak of the lowest concentration in the calibration curve, was 10 ng/ml (n = 6), with an accuracy of 105% and a coefficient of variation (CV) of 5.44%.For intraday analysis, six determinations of each QC concentration were assayed. For interday assessment, three replicates at each QC concentration were run on five separate days. The intra- and interday accuracies (percent bias) were within 11.0%. The intra- and interday precisions (CV) were within 6% (Table ​(Table1).1). The aqueous humor-based low (mean = 28.8 ng/ml; CV = 4.21%) and high (mean = 4,111 ng/ml; CV = 3.03%) QC samples gave results similar to those of the corresponding water-based QC samples. The retention times for caspofungin in aqueous humor- or water-based samples were identical.

TABLE 1.

Accuracy and precision data for assay of caspofungin

Development and Clinical Application of a New Method for the Radioimmunoassay of Arginine Vasopressin in Human Plasma

A radioimmunoassay has been developed that permits reliable measurements of plasma arginine vasopressin (AVP) at concentrations as low as 0.5 pg/ml in sample volumes of 1 ml or less. Nonhormonal immunoreactivity associated with the plasma proteins is eliminated by acetone precipitation before assay, leaving unaltered a component that is immunologically and chromatographically indistinguishable from standard AVP. Storage of plasma results in a decline in AVP concentration and, thus, must be carefully regulated. The plasma AVP values obtained by our method approximate the anticipated levels and vary in accordance with physiologic expections. In recumbent normal subjects, plasma AVP ranged from (mean ±SD) 5.4±3.4 pg/ml after fluid deprivation to 1.4±0.8 pg/ml after water loading, and correlated significantly with both plasma osmolality (r=0.52; P<0.001) and urine osmolality (r=0.77; P<0.001). After fluid restriction, plasma AVP was uniformly normal relative to plasma osmolality in patients with nephrogenic diabetes insipidus and primary polydipsia but was distinctly subnormal in all patients with pituitary diabetes insipidus. The infusion of physiologic amounts of posterior pituitary extract caused a dose-related rise in plasma vasopressin that afterwards declined at the expected rate (t½=22.5±4 min). We conclude that, when used appropriately, our radioimmunoassay method provides a useful way of assessing AVP function in man.     

高效液相色谱法在测定心肌组织中ATP、ADP、AMP方面的应用

目的：用高效液相色谱法来测定心肌组织中的ＡＴＰ、ＡＤＰ、ＡＭＰ的含量,比较含氧血心脏停搏液持续灌注和冷晶体心脏停搏间断灌注保护心肌的作用。方法：心脏瓣膜直视术患者４５例,随机分为两组冷晶体心脏停搏液组（ＣＣＣ组）和含氧血（Ｔ〉３５℃）心脏停搏液组（ＯＢＣ组）,分别测定两组心肌组织中ＡＴＰ、ＡＤＰ、ＡＭＰ的含量。结果：组间比较,主动脉开放后ＣＣＣ组能量储备值（ＥＣ值）明显降低（Ｐ〈０．０５）;组内比     

Validation and Pharmacokinetic Application of a High-Performance Liquid Chromatographic Technique for Determining the Concentrations of Amodiaquine and Its Metabolite in Plasma of Patients Treated with Oral Fixed-Dose Amodiaquine-Artesunate Combination in Areas of Malaria Endemicity

Artemisinin-based combination therapies (ACTs) have been adopted by most African countries, including Nigeria, as first-line treatments for uncomplicated falciparum malaria. Fixed-dose combinations of these ACTs, amodiaquine-artesunate (FDC AQAS) and artemether-lumefantrine (AL), were introduced in Nigeria to improve compliance and achieve positive outcomes of malaria treatment. In order to achieve clinical success with AQAS, we developed and validated a simple and sensitive high-performance liquid chromatography (HPLC) method with UV detection for determination of amodiaquine (AQ) and desethylamodiaquine (DAQ) in plasma using liquid-liquid extraction of the drugs with diethyl ether following protein precipitation with acetonitrile. Chromatographic separation was achieved using an Agilent Zorbax C₁₈ column and a mobile phase consisting of distilled water-methanol (80:20 [vol/vol]) with 2% (vol/vol) triethylamine, pH 2.2, at a flow rate of 1 ml/min. Calibration curves in spiked plasma were linear from 100 to 1,000 ng/ml (r > 0.99) for both AQ and DAQ. The limit of detection was 1 ng (sample size, 20 μl). The intra- and interday coefficients of variation at 150, 300, and 900 ng/ml ranged from 1.3 to 4.8%, and the biases were between 6.4 and 9.5%. The mean extraction recoveries of AQ and DAQ were 80.0% and 68.9%, respectively. The results for the pharmacokinetic parameters of DAQ following oral administration of FDC AQAS (612/200 mg) for 3 days in female and male patients with uncomplicated falciparum malaria showed that the maximum plasma concentrations (C_max) (740 ± 197 versus 767 ± 185 ng/ml), areas under the plasma concentration-time curve (AUC) (185,080 ± 20,813 versus 184,940 ± 16,370 h · ng/ml), and elimination half-life values (T_1/2) (212 ± 1.14 versus 214 ± 0.84 h) were similar (P > 0.05).