Determination of tacrolimus (FK 506) in whole blood using liquid chromatography and fluorescence detection

Summary A sensitive and selective liquid chromatographic method, using precolumn derivatization with dansylhydrazine followed by fluorescence detection, has been developed for the determination of tacrolimus (FK 506) in whole blood. After haemolysis, whole blood samples are extracted with diethyl ether and derivatized. After on-line removal of excess dansylhydrazine on a C₁₈ precolumn, the derivative is loaded on to a C₁₈ clean-up column, and a heart cut is subsequently transferred to a graphitized carbon column, where the final separation takes place. The method requires 1 ml of sample and has a limit of quantitation of 3 ng/ml. At the 15 ng/ml level the precision isca 10%, and the response is linear from the limit of quantitation toca 200 ng/ml of FK 506 in whole blood. The capacity of the method is 50 samples/day and about 1000 1-ml samples can be analyzed without changing either clean-up or separation column. Finally, the applicability of the method for therapeutic drug monitoring is shown.     

Determination of cyclosporin A in blood and plasma by column switching-HPLC after rapid sample preparation

A method for the determination of cyclosporin A in human whole blood and plasma is described which uses liquid chromatography with step gradient elution and a column switching technique. The chromatographic conditions chosen allow simple and rapid sample preparation, so that a result can be obtained within one hour. Blood and plasma are deproteinized with diluted methanol and an aliquot of the clear supernatant is directly injected. The detection limit for cyclosporin A is about 20 ng/ml starting from a 0.5 ml sample. The method is sensitive enough for monitoring the drug in the therapeutic range.     

Improved high-performance liquid chromatographic method for analysis of cyclosporin A using an automated sample processor

Transplant patients receiving the immunosuppressive drug cyclosporin A require regular monitoring to maintain levels within a narrow therapeutic range. A stable, accurate and reproducible high-performance liquid chromatographic method for analysis of cyclosporin A in whole blood has been developed using the Varian Advanced Automated Sample Processor. Starting with 200 microliters of blood, absolute recovery of both cyclosporin A and the internal standard was 81% with a detection limit of 12.5 ng/ml. The assay is perfectly linear over the range 0-1000 ng/ml (r2 = 1.0). At a concentration of 250 ng/ml, the coefficient of variation, both between samples and between assays, is 1.87%. Chromatographic cycle time is 10.2 min per sample. Up to eighty samples can be processed by one person in a working day, with final results within 16 h.     

Improvement of cyclosporin A determination in whole blood by reversed-phase high-performance liquid chromatography

A chromatographic method was developed for the determination of cyclosporin A in human whole blood using reversed-phase HPLC at room temperature. Most previous reports carried out this liquid chromatographic separation at temperatures above 70 degrees C. The present procedure greatly improves the detection limit by controlling peak broadening effects, as well as the lifetime of the column at room temperature. Under optimal conditions and using ketoconazole as an internal standard, the calibration graph was linear in the range of 16-1000 microg/L with a relative standard deviation of 3.72% at 150 microg/L and 2.45% at 300 microg/L (n = 11) of cyclosporin A. The detection limit was of 5.0 microg/L cyclosporin A. By this procedure, cyclosporin A pharmacokinetic parameters in healthy Chinese subjects were studied. The developed method could be applied to the quantification of cyclosporin A in human blood samples and allows the study of its pharmacokinetics in routine laboratories.     

Development of direct stereoselective and non-stereoselective assays in biological fluids for the enantiomers of a thieno[2,3-b]thiopyran-2-sulfonamide, a topically effective carbonic anhydrase inhibitor

A stereoselective assay for the optical isomers [(S) and (R)] of 5,6-dihydro-4-[(2-methylpropyl)amino]-4H-thieno[2,3-b]thiopyran-2- sulfonamide-7,7-dioxide in human whole blood has been developed. The assay is based on direct enantiomer separation on a chiral stationary phase column of bovine serum albumin attached to silica. The effect of pH, ionic strength, column length and organic modifier on chiral separation has been studied. The assay methodology, based on high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection (252 nm), has been fully validated in the concentration range 25-250 ng/ml of each enantiomer. Since no interconversion of the isomers was observed in vivo for the clinical studies involving the single (S)-enantiomer, a more sensitive (2.5 ng/ml), non-stereoselective assay has been developed. This method, also based on HPLC with UV detection, was fully validated in whole blood, plasma and urine in the concentration range 2.5-100 ng/ml. The details of these assays, together with some representative data from a pilot human study, are also presented.     

Semi-automated high-performance liquid chromatographic determination of cyclosporine A in whole blood using one-step sample purification and column-switching

A highly sensitive and semi-automated high-performance liquid chromatographic method, utilizing acetonitrile protein precipitation and column-switching, is described for the determination of cyclosporine A in whole blood. Following a rapid manual acetonitrile treatment of the blood samples, the supernatant is loaded automatically onto a 5-micron high-speed protein separation column without any further clean-up operations. The fraction containing cyclosporine A is switched to a 3-micron C18 reversed-phase high-speed column by a microprocessor-controlled column-switching unit for final separation and detection by absorption at 214 nm. Minimal sample handling and efficient separation resulted in a high recovery (75 +/- 3%) of cyclosporine A from blood and a detection limit as low as 2 micrograms/l with a highly reproducible and linear response up to 2500 micrograms/1 using 0.5 ml of sample. A separation cycle including regeneration of the first column is finished in 15 min, and this system was used continuously for ca. 1000 blood samples from heart, liver, kidney, pancreas and bone marrow recipients without change in separation parameters or material replacement. The method described allows accurate and very fast daily routine monitoring of cyclosporine A in large numbers of blood samples from transplant recipients.     

Natural analcime zeolite modified with 5-Br-PADAP for the preconcentration and anodic stripping voltammetric determination of trace amount of cadmium.

A procedure for separation and preconcentration of trace amounts of cadmium has been proposed. A column of analcime zeolite modified with benzyldimethyltetradecylammonium chloride and loaded with 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (5-Br-PADAP) was used for retention of cadmium. The cadmium was quantitatively retained on the column at pH approximately 9 and was recovered from column with 5 ml of 2 M nitric acid with a preconcentration factor of 140. Anodic stripping differential pulse voltammetry was used for determination of cadmium. A 0.05 ng/ml detection limit for the preconcentration of aqueous solution of cadmium was obtained. The relative standard deviation (RSD) for eight replicate determinations at the 1 microg/ml cadmium levels was 0.31% (calculated with the peak height obtained). The calibration graph using the preconcentration system was linear from 0.01 to 150 microg/ml in final solution with a correlation coefficient of 0.9997. For optimization of conditions, various parameters such as the effect of pH, flow rate, instrumental conditions and interference of number of ions, were studied in detail. This method was successfully applied for determination of cadmium in various complex samples.     

高效液相色谱法快速同时检测全血中两种免疫抑制剂

环孢素A和西罗莫司是许多器官移植手术中广泛使用的免疫抑制剂,且一起使用时会产生协同效应,但这两种免疫抑制剂的治疗窗口都非常窄,仅在特定的血药浓度范围内有预期的治疗效果。因此,快速同时检测全血中这两种免疫抑制剂的浓度,可为患者器官移植手术后的给药方案提供有价值的信息。该工作首先考察了环孢素A和西罗莫司在生物液相色谱柱和传统液相色谱柱上的色谱行为,然后基于生物液相色谱柱,建立了可快速分离和检测全血中环孢素A和西罗莫司的高效液相色谱分析方法。全血样品经样品前处理后进样分析,采用ZORBAX 300SB C8柱（250 mm×4.6 mm, 5.0 μm）进行分离,以乙腈-水（70∶30, v/v）为流动相进行等度洗脱,柱温为60 ℃,流速为1.0 mL/min,检测波长为205 nm和278 nm,进样量为20 μL。结果表明,环孢素A和西罗莫司在6 min内可实现较好的分离;环孢素A和西罗莫司在各自的浓度范围内具有良好的线性关系（r>0.997）,检出限（S/N=3）分别为10 ng/mL和1 ng/mL,定量限（S/N=10）分别为30 ng/mL和2 ng/mL, 3个水平的平均加标回收率分别为83.5%~89.7%和95.8%~97.8%,相对标准偏差（RSD）分别为3.2%~9.0%和3.4%~6.7%（n=5）。该方法操作简便,流动相简单,分析时间短,线性范围宽,灵敏度高,可用于全血中环孢素A和西罗莫司的含量检测。     

Rapid determination of clenbuterol residues in urine by high-performance liquid chromatography with on-line automated sample processing using immunoaffinity chromatography

A liquid chromatographic column-switching system for automated sample pretreatment and determination of clenbuterol in calf urine, using an immunoaffinity precolumn with Sepharose-immobilized polyclonal antibodies against clenbuterol, is described. A second precolumn packed with C18-bonded silica was used for the reconcentration of desorbed clenbuterol prior to the analytical separation. Urine, after 2-fold dilution with buffer (pH 7.4), was loaded directly onto the immuno precolumn, where clenbuterol was trapped by the immobilized antibodies. This immuno precolumn has been used for more than 200 runs with standard solutions and samples. Bound analyte was desorbed with 0.01 M acetic acid and transferred, via the second precolumn, to the analytical column. The total runtime per sample was 35 min. Using a sample load of 27 ml of dilute urine and UV detection at 244 nm, the detection limit was 0.5 ng/ml. The mean recovery of clenbuterol added to a blank urine sample at the 5 ng/ml level was 82 +/- 2% (n = 5) as determined with standard solutions loaded onto the same system. Urine samples from treated animals were analysed and the clenbuterol concentrations were comparable to those obtained by high-performance liquid chromatography using solid-phase extraction for sample clean-up.     

Determination of nonylphenol polyethoxylates in household detergents by high-performance liquid chromatography

The binding of metals to proteins in blood fractions was investigated applying hydrophobic interaction chromatography (HIC) for protein separation and graphite furnace atomic absorption spectrometry (GFAAS) as the element specific detector. For the semi-preparative separation of metalloproteins in erythrocytes and blood plasma, a HIC column (Fractogel EMD Phenyl I (S) 150 mm×10 mm I.D.) was adapted. The separation column was calibrated with the same four standard proteins as used in Pomazal et al. [Analyst 124 (1999) 657]. The sample injection volume and the ammonium sulphate gradient set-up were optimized: 20 or 200 μl, respectively, of blood plasma and of lysed erythrocytes were injected. The separated proteins were collected in 4-ml fractions and analyzed by GFAAS off-line. An optimization of the GFAAS measuring parameters for Cu, Mn, Fe, Zn, Co, Ni, and Cr was performed. For each element, a specific temperature program was optimized with respect to the matrix of the HIC eluate (0.02 M NaH₂PO₄, 1.8 M (NH₄)₂SO₄). The obtained metal profiles of the eluate were compared with the HIC chromatograms. The limits of detection (LOD) for the elements by GFAAS were: 0.5 ng Cu/ml; 0.2 ng Mn/ml; 1 ng Fe/ml; 0.2 ng Zn/ml; 0.12 ng Co/ml; 0.2 ng Ni/ml; 0.16 ng Cr/ml. The GFAAS method enabled the detection of the proteins of interest via the metals.     

Determination of cadralazine in human whole blood using reversed-phase high-performance liquid chromatography: utilizing a salting-out extraction procedure

A rapid, selective, and sensitive ion-paired reversed-phase high-performance liquid chromatographic method for determination of the new carbazate type of antihypertensive vasodilator agent cadralazine in human whole blood has been developed. Cadralazine was extracted from whole blood by adding 0.5 ml of acetonitrile to 1.0 ml of whole blood followed by salting-out of acetonitrile by the addition of potassium carbonate in excess. An aliquot of the salted-out acetonitrile was injected into the chromatographic system. A column packed with 3-microns octyl (C8) particles was used with an isocratic elution of 1% acetic acid and 5 mM hexanesulfonic acid-acetonitrile (70:30, v/v). The cadralazine was measured using ultraviolet detection at 250 nm and the assay was completed in less than 20 min and had a limit of quantitation of 10 ng/ml for a 100-microliters injection volume.     

Fully automated high-performance liquid chromatographic analysis of whole blood and plasma samples using on-line dialysis as sample preparation. Determination of oxytetracycline in bovine and salmon whole blood and plasma.

A fully automated technique for high-performance liquid chromatographic analysis of whole blood and plasma is described. Samples are automatically injected into a dialyser where proteins and blood cells are removed. The dialysates are concentrated on a small column prior to analysis. This technique is used for the determination of oxytetracycline in whole blood and plasma. After dialysis oxytetracycline and the internal standard, tetracycline, are retained on a polystyrene enrichment column and subsequently separated on a polystyrene analytical column by ion-pair chromatography. Using ultraviolet detection 50 ng/ml can be detected. Validation showed good within-day and between-day accuracy and precision. Different oxytetracycline concentrations were found in plasma and whole blood. This difference varied between the species.     

Analytical and semi-preparative high-performance liquid chromatographic separation and assay of hydroxychloroquine enantiomers.

(+/-)-Hydroxychloroquine (HCQ) is an antimalarial and anti-arthritic drug which is administered as the racemate. An accurate, precise and sensitive high-performance liquid chromatographic assay was developed for the determination of HCQ enantiomers in samples from human plasma, serum, whole blood, and urine. After addition of (+/-)-chloroquine (internal standard), samples of blood component (0.5 ml) or urine (0.1 ml) were alkalinized and extracted with 5 ml of diethyl ether. After solvent evaporation the residues were derivatized with (+)-di-O-acetyl-L-tartaric anhydride at 45 degrees C for 30 min. The resulting diastereomers were then resolved using a C8 analytical column with a mobile phase consisting of 0.05 M KH2PO4 (pH 3)-methanol-ethanol-triethylamine (78:22:1:0.08). The ultraviolet detection wavelength was set at 343 nm. The derivatized HCQ enantiomers eluted in less than 40 min, free of interfering peaks. Excellent linear relationships (r2 > 0.997) were obtained between the area ratios and the corresponding plasma concentrations over a range of 12.5-500 ng/ml. The diastereomers could be hydrolysed using microwave energy and neutral pH, which enabled us to resolve the enantiomers on a semi-preparative (C18 column) scale. The method was suitable for the analysis and semi-preparative separation of HCQ enantiomers.     

Quantitation of Verapamil and Norverapamil in Small Blood Samples From the Rat by High Performance Liquid Chromatography

Abstract

A simple, sensitive HPLC assay using flurescence detection was developed for quantitation of verapamil and its active metabolite, norverapamil in 100-200 μl blood samples from the rat. Baseline separation of verapamil, normverapamil and internal standard, propranolol, was attained within 14 minutes. Standard curves for verapamil and norverapamil were linear from 7 ng/ml to 1000 ng/ml with limit of detection of 4 ng/ml for both Compounds. the intraday and interday coefficients of variation in verapamil and norverapamil concentrations, determined from spiked whole blood samples, were less than 10%.     

An HPLC method with diode array detector for the simultaneous quantification of chloroquine and desethylchloroquine in plasma and whole blood samples from Plasmodium vivax patients in Vietnam,using quinine as an internal standard

A sensitive, simple method for quantification of chloroquine (CQ) and desethylchloroquine (MCQ) in whole blood and plasma from Plasmodium vivax patients has been developed using HPLC with diode array detection (DAD). Solid‐phase extraction on Isolute‐96‐CBA was employed to process 100 μL of plasma/whole blood samples. CQ, MCQ and quinine were separated using a mobile phase of phosphate buffer 25 mm , pH 2.60–acetonitrile (88:12, v/v) with 2 mm sodium perchlorate on a Zorbax SB‐CN 150 × 4.6 mm, 5 μm column at a flow rate of 1.2 mL/min, at ambient temperature in 10 min, with the DAD wavelength of 343 nm. The method was linear over the range of 10–5000 ng/mL for both CQ and MCQ in plasma and whole blood. The limit of detection was 4 ng/mL and limit of quantification was 10 ng/mL in both plasma and blood for CQ and MCQ. The intra‐, inter‐ and total assay precision were <10% for CQ and MCQ in plasma and whole blood. In plasma, the accuracies varied between 101 and 103%, whereas in whole blood, the accuracies ranged from 97.0 to 102% for CQ and MCQ. The method is an ideal technique with simple facilities and instruments, bringing about good separation in comparison with previous methods. © 2016 The Authors Biomedical Chromatography Published by John Wiley & Sons Ltd     

Reversed-phase liquid chromatographic determination of idarubicin and its 13-hydroxy metabolite in human plasma

A method is given for the determination of idarubicin and its main metabolite, idarubicinol, in plasma from cancer patients. Idarubicin and idarubicinol are extracted from 2-ml samples of buffered plasma (pH 8.1) using chloroform-1-heptanol (9:1). After reextraction into phosphoric acid (0.1 M), separation is performed by reversed-phase liquid chromatography on a LiChrosorb RP-2 column (5 microns) with a mobile phase of acetonitrile-water, acidified with phosphoric acid. The absolute recovery in the range 5-100 ng/ml was greater than 83% with a precision better than 8% (relative standard deviation), using photometric detection at 484 nm. Proper handling of whole blood samples containing idarubidin is essential to avoid metabolic conversion into idarubicinol. Prolonged storage of the drug and its main metabolite under alkaline conditions should be avoided to prevent chemical degradation.     

Rapid extraction of codeine and morphine in whole blood for HPLC analysis

Summary A rapid and efficient procedure is described for the extraction and analysis of codeine and morphine in whole blood. Red blood cells were fragmented by sonication and the blood sample extracted by passing through a bonded silica column (Bond Elute^?). The adsorbed drugs were washed and eluted followed by analysis by HPLC. Recoveries were between 95–100% at 5 ng/ml concentrations.     

Determination of noscapine in plasma by liquid chromatography

A liquid chromatographic method has been developed for the determination of noscapine in plasma. Noscapine and the internal standard, papaverine, were extracted into methylene chloride by column extraction. The separation was performed on a straight-phase liquid chromatographic system using a mobile phase of hexane--methanol--chloroform--diethylamine. A high detection selectivity was obtained by UV detection at 310 nm. The precision of the method was 3.8% (standard deviation) at a level of 89 ng/ml and 9.5% (standard deviation) at 5.9 ng/ml. The selectivity of the analytical method was evaluated by comparing analytical results after isolation of extracts of plasma samples on reversed- and straight-phase liquid chromatographic systems.     

Determination of lansoprazole and its metabolites in plasma by high-performance liquid chromatography using a loop column.

A high-performance liquid chromatographic method for the simultaneous determination of lansoprazole, a new proton pump inhibitor, and its metabolites in human plasma is described. Lansoprazole, its metabolites and an internal standard were extracted with tert.-butyl methyl ether. Samples were injected using an automatic injector via a loop column, and separation was obtained using a reversed-phase column under isocratic conditions. The absorbance was monitored at 285 and 303 nm. The quantification limit was 2 ng/ml for lansoprazole and 3 or 5 ng/ml for the metabolites. No endogenous compounds were found to interfere. The mean overall recovery was between 75 and 95% for lansoprazole and its metabolites. This method is suitable for pharmacokinetic studies.     

Method for quantification of morphine and its 3- and 6- glucuronides, codeine, codeine glucuronide and 6-monoacetylmorphine in human blood by liquid chromatography-electrospray mass spectrometry for routine analysis in forensic toxicology.

Simultaneous determination of opiates and their glucuronides in body fluids has a great practical interest in the forensic assessment of heroin intoxication. A selective and sensitive method for quantification of morphine and its 3- and 6-glucuronides, codeine, codeine glucuronide and 6-monoacetylmorphine (6-MAM) based on liquid chromatography-electrospray ionisation mass spectrometry is described. The drugs were analysed in human autopsy whole blood after solid-phase extraction on a C8 cartridge. The separation was performed on an ODS column in acetonitrile (analysis time 15 min). For the quantitative analysis, deuterated analogues of each compound were used as internal standards. Selected-ion monitoring was applied where the molecular ion was chosen for quantification. The limits of quantification were 0.5 ng/ml for morphine and 6-MAM and 1 ng/ml for the 6-glucuronide of morphine, codeine-6-glucuronide and codeine and 5 ng/ml for the 3-glucuronide of morphine.