Anidulafungin—challenges in development and validation of an LC-MS/MS bioanalytical method validated for regulated clinical studies

Anidulafungin is a semi-synthetic echinocandin with antifungal activity, usually administered as an intravenous infusion. In order to determine the pharmacokinetics (PK) of anidulafungin in pediatric patients, a sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) bioanalytical method (M1) was developed and validated for quantification of anidulafungin in plasma. During analysis of incurred samples (samples collected from patients enrolled in a clinical study) an isobaric chromatographic interference was observed. The source of interference was identified as an anidulafungin open-ring form (D1) and its impact on the quantification of anidulafungin was investigated. It was found that accurately quantifying anidulafungin in incurred samples required chromatographic separation of the open-ring form from anidulafungin. The method was redeveloped to achieve the appropriate baseline separation and to avoid experimental conditions that favored opening the anidulafungin ring. The extraction of anidulafungin from plasma by protein precipitation remained unchanged, but the changes in chromatography warranted validation of a new method, M2, 2?years after M1 was validated. Incurred samples from three studies that were previously analyzed by M1 and were within confirmed long-term frozen stability were then reanalyzed by M2. Although the incurred sample reproducibility tests on those samples passed for each of the two methods, comparison of concentrations from the same samples obtained by M1 and M2 revealed that an overestimation of anidulafungin following the M1 method exceeded acceptance criteria. The new HPLC-MS/MS method (M2) is applicable for quantification of anidulafungin within a nominal range 50-20,000?ng/mL and requires a 50?μL human plasma aliquot. A linear, 1/concentration squared weighted, least-squares regression algorithm was used to generate the calibration curve and its parameters were used to quantitate the incurred samples. The inter-assay accuracy in heparin human plasma validation ranged from -4.33 to 0.0386?% and precision was ≤7.32?%. The method M2 was validated for use in regulated bioanalysis and is presently used to quantitate anidulafungin in plasma samples from clinical studies.     

Determination of 3'-deamino-3'-[2(S)-methoxy-4-morpholinyl]doxorubicin, a new morpholinyl anthracycline, in plasma by high-performance liquid chromatography with fluorescence detection.

A sensitive and selective high-performance liquid chromatographic method for the determination of 3'-deamino-3'-[2(S)-methoxy-4-morpholinyl]doxorubicin and its possible 13-dihydro metabolite in human plasma has been developed. The plasma samples were buffered and the drugs and internal standard (doxorubicin) were extracted with diethyl ether-n-butanol, back-extracted into 0.3 M phosphoric acid, then analysed by reversed-phase liquid chromatography. Quantitation was achieved by fluorescence detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from blank plasma sample was observed. The suitability of the method for in vivo samples was checked by analysis of plasma samples drawn from female rats that had received repeated intravenous doses of the test compound.     

反相高效液相法测定血浆及尿液中的异烟肼

 建立了血浆及尿液中异烟肼的高效液相快速测定方法 ,以满足临床药物分析和法医学鉴定的需要 ,提高对血浆及尿液中异烟肼浓度检测的准确性。以香草醛为衍生化试剂 ,将异烟肼经柱前衍生为异烟肼 香草醛腙 ,直接对处理后样品中的腙进行定性、定量分析。以在空白人体液样本中定量添加标准异烟肼的方法考察了样品的前处理方法、仪器条件、线性范围、精密度、回收率等 ,并对健康受试者血液中的异烟肼浓度进行了监测。结果表明 ,方法的线性范围为 0 2mg/L～ 1 2 0mg/L ;检测限为 0 2mg/L ;日内、日间精度均小于 4 0 % (n =5) ;回收率在96 3 %以上。     

液相色谱-串联质谱法测定生物样本全基因组DNA甲基化

建立了基于液相色谱-电喷雾串联质谱的分析方法,对生物样本中全基因组DNA甲基化水平进行定量测定.首先将DNA从生物样本中提取出来,将DNA片段酶解为单核苷,利用液相色谱-串联质谱测定胞嘧啶核苷和5-甲基胞嘧啶核苷的含量,从而计算出其全基因组DNA甲基化率.利用该法研究了暴露于全氟辛烷磺酸的L-02细胞、10例原发性肝癌病例血浆样本和10例对照血浆样本的全基因组DNA甲基化水平,得出了它们的总甲基化率变化的初步结果.本方法操作简单,具有很高的灵敏度和稳定性,为研究生物样本,尤其是临床上易得但DNA含量极低的血浆样本的总甲基化水平提供了思路.     

Capillary electrophoresis analysis of fosfomycin in biological fluids for clinical pharmacokinetic studies

A feasible capillary zone electrophoresis (CZE) method with indirect UV and contactless conductivity detection was developed for the determination of fosfomycin, an antibiotic, in human plasma and microdialysis samples. Samples were collected from test persons during a clinical trial. The background electrolytes used consisted of 25 mM benzoic acid and 0.5 mM hexadecyltrimethylammonium bromide, adjusted with tris(hydroxymethyl)aminomethane solution to pH 6.95 for plasma, and to pH 8.05 for microdialysis samples. CZE separations of the anionic analyte were carried out with reversed electroosmotic flow directed towards the anode. The limit of detection was between 0.6 and 2 microg/mL, depending on the matrix and the detection method. No sample preparation was needed for microdialysis samples; for plasma samples, proteins were precipitated with methanol (1+2, v+v), and the supernatant was analyzed. The yield determined with spiked samples was about 100%, the reproducibility of the entire method, expressed by the RSD% of three independent determinations of fosfomycin in triplicate after spiking Ringer's solutions and plasma samples, respectively, was better than 8%. The method is thus well-suited for clinical studies for the determination of the antibiotic in biological fluids.     

Quantitative determination of metformin,glyburide and its metabolites in plasma and urine of pregnant patients by LC‐MS/MS

This report describes the development and validation of an LC‐MS/MS method for the quantitative determination of glyburide (GLB), its five metabolites (M1, M2a, M2b, M3 and M4) and metformin (MET) in plasma and urine of pregnant patients under treatment with a combination of the two medications. The extraction recovery of the analytes from plasma samples was 87–99%, and that from urine samples was 85–95%. The differences in retention times among the analytes and the wide range of the concentrations of the medications and their metabolites in plasma and urine patient samples required the development of three LC methods. The lower limit of quantitation (LLOQ) of the analytes in plasma samples was as follows: GLB, 1.02 ng/mL; its five metabolites, 0.100–0.113 ng/mL; and MET, 4.95 ng/mL. The LLOQ in urine samples was 0.0594 ng/mL for GLB, 0.984–1.02 ng/mL for its five metabolites and 30.0 µg/mL for MET. The relative deviation of this method was <14% for intra‐day and inter‐day assays in plasma and urine samples, and the accuracy was 86–114% in plasma, and 94–105% in urine. The method described in this report was successfully utilized for determining the concentrations of the two medications in patient plasma and urine. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of trichlormethiazide in human plasma and urine by high-performance liquid chromatography

Summary This paper describes a high-performance liquid chromatographic (HPLC) assay method for the determination of trichlormethiazide (TCM) in human plasma and urine. After extraction and separation on an ODS column TCM from plasma was detected by oxidation in an electrochemical detector (ECD) by a porous graphite electrode. The sensitivity was better than HPLC with UV detection, enabling the determination of 2 ng ml^–1 TCM in human plasma. This method also allows determination of TCM at higher concentrations by exchanging the UV for the electrochemical detector. To study the pharmacokinetics, TCM in plasma and urine was assayed with coefficients of variation in the range 2–3%. The method has the advantages of high sensitivity for plasma assay and high precision with a simple procedure for both plasma and urine samples. Small samples of 0.5 ml plasma per assay also reduced the total volume of plasma needed.     

High performance liquid chromatography with UV detection for the simultaneous determination of sympathomimetic amines using 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride as a label

A high performance liquid chromatographic method has been developed for the simultaneous determination of (+/-) fenfluramine (Fen) and phentermine (Phen) in addition to three other sympathomimetic amines-ephedrine (E), norephedrine (NE) and 2-phenylethylamine (2-PEA), using cyclohexylamine (CX) as an internal standard in plasma. The compounds were derivatized with 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride (DIB-Cl) to give the DIB-derivatives. The derivatives were then separated using an isocratic HPLC system with UV detection. The limits of detection for Fen, Phen, E, NE and 2-PEA in plasma ranged from 0.32 to 22.9 pmol on column at a signal-to-noise ratio of 3. The recoveries following alkaline extraction from plasma samples of known concentrations were found to be more than 94% for the studied compounds. This method might be useful for the screening of the studied sympathomimetic amines in human plasma samples in forensic as well as toxicological studies. Furthermore, the developed method was modified for the simultaneous determination of Fen and Phen in human and rat plasma using fluoxetine as an internal standard. The methods are reproducible and precise. Finally, the two drugs were administered intraperitoneally to rats in combination, and their plasma levels over the investigated time course were successfully determined.     

Simple determination of forphenicinol in human plasma and erythrocytes by HPLC with native fluorescence detection

A high performance liquid chromatographic method is described for monitoring forphenicinol, a possible therapeutic drug for cancer and muscular dystrophy, in human plasma and erythrocytes. Forphenicinol in the deproteinized samples was separated from interfering biogenic substances on an aminopropyl-bonded silica (Unicil NH2) column within 10 minutes with isocratic elution, and determined with fluorescence detection. The detection limits for forphenicinol in plasma and erythrocytes are 65 pmol (12.8 ng)/ml and 160 pmol (31.5 ng)/ml, respectively, corresponding to 2 pmol each in a 100 microliters injection volume. The method is very simple, and sensitive enough to permit the quantification of forphenicinol in the blood samples from man dosed with forphenicinol.     

HPLC with diode-array detection for the simultaneous determination of di(2-ethylhexyl)phthalate and mono(2-ethylhexyl)phthalate in seminal plasma

A high-performance liquid chromatographic method for the simultaneous determination of di(2-ethylhexyl)phthalate (DEHP) and its major metabolite mono(2-ethylhexyl)phthalate (MEHP) in seminal plasma was developed and validated. The method involves liquid-liquid extraction followed by isocratic reversed-phase chromatography with diode-array detection. The recovery, selectivity, linearity, precision and accuracy of the method were evaluated from the analysis of spiked seminal plasma samples. The effect of mobile-phase composition and pH on the retention of the target analytes was investigated. The limits of detection were 0.010 and 0.015 microg/mL, for DEHP and MEHP, respectively. This method was used to analyze real samples in support of clinical studies on these potential endocrine disruptors.     

Determination of carbocysteine in human plasma by liquid chromatography/tandem mass spectrometry employing precolumn derivatization

A sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed to determine carbocysteine in human plasma using 2-pyridylacetic acid as the internal standard (IS). The method employed derivatization with 10 M hydrochloric acid/methanol, which significantly improved the ionization efficiency of carbocysteine. After methanol-induced protein precipitation of plasma samples, carbocysteine and the IS were derivatized and subjected to LC/MS/MS analysis using atmospheric pressure chemical ionization. The method has a lower limit of quantitation of 20 ng/mL for a 0.2-mL plasma aliquot. The intra- and inter-day precision (RSD), calculated from quality control (QC) samples, was less than 7%. The accuracy, determined using QC samples, was within +/- 1%. The method offered increased sensitivity, selectivity and speed of analysis over existing methods. The method was utilized to support clinical pharmacokinetic studies of carbocysteine in volunteers following oral administration.     

An integrated bioanalytical method development and validation approach: case studies

We proposed an integrated bioanalytical method development and validation approach: (1) method screening based on analyte's physicochemical properties and metabolism information to determine the most appropriate extraction/analysis conditions; (2) preliminary stability evaluation using both quality control and incurred samples to establish sample collection, storage and processing conditions; (3) mock validation to examine method accuracy and precision and incurred sample reproducibility; and (4) method validation to confirm the results obtained during method development. This integrated approach was applied to the determination of compound I in rat plasma and compound II in rat and dog plasma. The effectiveness of the approach was demonstrated by the superior quality of three method validations: (1) a zero run failure rate; (2) >93% of quality control results within 10% of nominal values; and (3) 99% incurred sample within 9.2% of the original values. In addition, rat and dog plasma methods for compound II were successfully applied to analyze more than 900 plasma samples obtained from Investigational New Drug (IND) toxicology studies in rats and dogs with near perfect results: (1) a zero run failure rate; (2) excellent accuracy and precision for standards and quality controls; and (3) 98% incurred samples within 15% of the original values.     

Analysis of norethindrone in plasma by high-performance liquid chromatography

The lack of specificity of some radioimmunological assays for the determination of norethindrone has been reported. This paper describes a high performance liquid chromatographic (HPLC) method that is sufficiently sensitive and specific for the determination of plasma samples containing 2 ng/ml norethindrone. Plasma samples were collected from 8 human volunteers. The solvents used for the mobile phase were methanol, HPLC grade acetonitrile, HPLC grade, and double glass-distilled water. The HPLC (Waters Associates, Milford, Massachusetts) was used at a nominal attenuation of .005 absorbance units full scale (AUFS) and fed into a 5 mV recorder, giving an overall response of 0.0025 AUFS. The HPLC was also fitted with an ultraviolet detector (254 nm). The organic solvent extract from plasma is chromatographed on a reversed phase column using the HPLC. Tritiated norethindrone was added to 2.0 ml of plasma containing from 2 to 20 ng/ml of norethindrone and was extracted using the procedure as described. The organic extract was then transferred into a scintillation vial and evaporated to dryness. A Beckman LS 150 scintillation counter with an automatic quench correction device was used to determine radioactivity. The results show that the extraction efficiency and reproducibility are comparable at plasma concentrations ranging between 2-20 ng/ml. No interfering compounds (metabolites and endogenous substances) were extracted using HPLC. Data analysis of a number of spiked plasma samples ranging from 2 ng/ml-20 ng/ml suggests the accuracy and precision of the method. In another experiment, 40 mg of norethindrone was dissolved in ethanolic saline and orally administered in a mini-pig. The plasma norethindrone concentration was readily detected. The experiments illustrate the stronger specificity of the new method compared to reported radioimmunoassays.     

Application of microcrystalline cellulose as an efficient and cheap sorbent for the extraction of metoprolol from plasma and wastewater before HPLC–MS/MS determination

A dispersive solid-phase extraction method based on a new sorbent has been performed on plasma and wastewater samples to determine metoprolol by high-performance liquid chromatography–tandem mass spectrometry. In this study, the analyte was adsorbed from the samples onto microcrystalline cellulose as a green and efficient sorbent and then eluted for use in the determination step. In the mass spectrometer, the analyte was detected in the positive mode and selectivity of the analysis was increased by sequential mass analysis through multiple reaction monitoring. All of the effective parameters in the extraction of metoprolol from plasma and wastewater were optimized. Under optimal conditions the method was linear in the ranges of 1–1,000 and 0.1–1,000 ng/ml in plasma and wastewater samples, respectively. The detection limits of the method were 0.30 and 0.03 ng/ml in plasma and wastewater samples, respectively. The data showed that the method provides low detection limit, wide linear range, good precision and high extraction recovery. Finally several plasma and wastewater samples were successfully analyzed using the method. The use of a small amount of a green and inexpensive sorbent and a low volume of plasma without the need for further pretreatment steps are the main advantages of the method.     

Determination of 2′,3′-Dideoxyinosine in Plasma by High Performance Liquid Chromatography

Abstract

A high performance liquid chromatography analysis method has been developed for the quantitation of 2′,3′-dideoxyinosine (DDI) in plasma. Proteins were precipitated from plasma samples with acetonitrile containing the internal standard, 6-methylaminopurine riboside. The treated samples were evaporated to dryness and reconstituted in mobile phase for the analysis. Separation of the components was achieved on a 5 μm octadecylsilane column with ultraviolet detection at 254 nm. The method was validated at nine concentrations between 0.015 and 150 μg/mL. Using 500 μL of human plasma, the limit of quantitation was 120 ng/mL and the limit of detection was 60 ng/mL. The mean intra-day precision of the method was 1.6%. The mean accuracy of the method was within 2% of the actual values. This method is currently being used for pharmacokinetic studies in the rat.     

三维荧光二阶校正方法用于体液和细胞培养基中五味子甲素含量的快速测定

采用三维激发发射荧光光谱结合自加权交替三线性分解(SWATLD)二阶校正方法, 对人体液样(血浆样及尿液样)和细胞培养基样中五味子甲素的含量进行了直接快速定量分析. 在血浆背景、尿液背景和细胞培养基背景共存下, 当分析体系的组分数分别选择2时, 用SWATLD二阶校正方法获得相应五味子甲素的平均回收率分别为(100.4±1.6)%, (100.5±6.3)%和(103.6±4.5)%. 实验结果表明, 此方法不仅能够较好地解决这些复杂分析体系因背景内源荧光性物质与待分析物光谱严重重叠所引起的难分辨的问题, 还可以用于直接快速准确定量分析.     

Synthesis,characterization, and application of griseofulvin surface molecularly imprinted polymers as the selective solid phase extraction sorbent in rat plasma samples

In present study, an investigation was carried out to develop and validate an analytical method for the selective extraction and determination of griseofulvin (GSF) from plasma samples. For this purpose, a rational approach was made to synthesize and characterize the surface molecularly imprinted polymers (SMIPs). The SMIPs were utilized as solid phase extraction (SPE) sorbents. The SMIPs were prepared by using GSF as template molecule on the surface of modified silica particles through a non-covalent technique. The particles demonstrated high adsorption capacity (119.1 µg/mL), fast adsorption equilibrium time (30 min) and good recognition selectivity for the template drug. The scanning electron microscopy and infrared spectroscopy were used to explain the structural and morphological characteristics of the SMIPs and surface non-imprinted polymers. The SPE method was combined with HPLC for plasma analysis. The method validation results demonstrated that the established method possessed good linearity for GSF ranging from 0.1 to 50 µg/mL (R² = 0.997). The limit of detection for this method was 0.02 µg/mL for rat plasma samples. The recoveries of GSF from spiked plasma samples were (90.7–97.7%) and relative standard deviations were (0.9–4.5%). Moreover, the SMIPs as selective SPE sorbent can be reused more than 8 times which is a clear advantage over commercial SPE sorbents. Finally, the usefulness of the proposed strategy was assessed by extraction and detection of GSF in real rat plasma samples.     

Validation and application of an HPLC method for determination of di (2-ethylhexyl) phthalate and mono (2-ethylhexyl) phthalate in liver samples

A reversed-phase gradient elution, UV detection method is developed for the simultaneous determination of mono (2-ethylhexyl) phthalate [MEHP] and di (2-ethylhexyl) phthalate [DEHP] in tissue samples. The method is validated with respect to extraction recovery, inter and intra-day precision, linearity of response, detect ability, and specificity. The validated method has been successfully applied to the study of DEHP and MEHP in liver, kidney, testis, brain, and plasma samples from rat.     

High-performance liquid chromatographic determination of lorazepam in monkey plasma

A simple, sensitive and selective method for the determination of lorazepam in monkey plasma has been developed using high-performance liquid chromatography in a reversed-phase mode. The limit of detection for lorazepam in plasma is about 2 ng/ml. The method has been applied to plasma samples obtained from cynomolgus monkeys after oral doses of 0.15 mg/kg and intravenous doses of 0.05 mg/kg of lorazepam. In this species, mean peak plasma concentrations of 12 ng/ml occurred at 2 h after oral dosing and declined with a half-life of 2,5 h; the mean terminal half-life after intravenous dosing was 1.4 h.     

Dispersive solid-phase extraction of risperidone from plasma samples using graphene oxide aerogels and determination with liquid chromatography

A graphene oxide-based aerogel was synthesized and applied to the extraction and the determinations with the high-performance liquid chromatography-ultraviolet detector. After the characterization of the produced graphene-aerogel, it was utilized as a dispersive solid-phase extraction sorbent for risperidone extraction from plasma samples. Aerogels are materials with a large surface area-to-mass ratio and plenty of core with functional groups which can easily attach to the analytes to extract them to the second phase. The suggested method determined risperidone in plasma samples in the wide dynamic range from 20 ng/ml to 3 μg/ml. The limits of detection and quantification of the developed method were calculated as 2.4 and 8.2 ng/ml, respectively. As a novel feature, the developed method has no need to precipitate plasma proteins, improving the analytical performance of the analysis. Also, for the first time, the produced materials were utilized for the extraction of risperidone from the plasma samples. The obtained results revealed that the developed approach could be employed as an accurate method for the quantification of risperidone in real plasma samples.