HPLC Measurement of Cyclosporine in Blood Plasma and Urine and Simultaneous Measurement of its Four Metabolites in Blood

Abstract

A high performance liquid chromatographic assay has been developed for the estimation of cyclosporine and its four major metabolites in blood and for cyclosporine alone in plasma and urine samples. This assay employs a rapid and very reproducible solid-liquid extraction system. Isocratic chromatographic conditions allow the simultaneous measurement of cyclosporine and its four major metabolites in blood. The method is linear up to 2500 ng/ml and the minimum quantifiable limit for cyclosporine is 30 ng/ml, when 1 ml of sample is analyzed.     

A Rapid Method For Cyclosporine A Determination By Hplc

Abstract

A rapid method is described for the determination of cyclo-sporine in whole blood by HPLC. The cyclosporine is extracted with an acetonitrile-isopropanol mixture, purified on a C₁₈ minicolumn, and finally injected on a CN column. Recovery of the drug is greater than 90%.     

Evaluation of a New Automated Method for Cyclosporine a Therapeutic Monitoring on Tdx Abbott

Abstract

A new automated method for measuring cyclosporine in plasma has been introduced by Abbott Laboratories, involving the TDx instrumentation and fluorescence polarization immunoassay. We have used currently the Sandoz Ciclosporin RIA for cyclosporine therapeutic monitoring for a long time. In order to find a method that will decrease the labor requirements associated with RIA procedure, we used specimens from 64

transplant patients to compare, both from analytical and clinical points of view, the Abbott assay with the Sandoz Ciclosporin assay. We believe that the Abbott assay offers some analytical advantages over the ciclosporin RIA procedure and can be used with confidence for cyclosporine therapeutic monitoring for kidney, heart and allogeneic bone marrow transplantation.     

Liquid Chromatographic Determination of Cyclosporine-A in Blood,with Chromosorb P Columns Used for Sample Purification

Abstract

A fast liquid-chromatographic determination of whole-blood cyclosporine A concenration is described. A sample preparation consisting of diethyl ether extraction and Chromosorb P column purification of extract, requires 1 mL of whole blood and takes 10 min of technical effort per sample. A reversed-phase C₁ column (5-μm particles) is used, with acetonitrile/methanol/water (20/45/35 by vol) as mobile phase. Cyclosporine A is quantified by absorbance at 214 nm, with cyclosporine D as internal standard. Chromatographic development is complete in 8 min. Linearity is verified by a five-point calibration curve in the range 50–900 μg/L, correlation coefficient r>0.998, y = 0.001x ? 0.053. Lower limit of sensitivity is 25 μg/L. Extraction efficiency was over 70%, accuracy varied between ?7.1% and +3.3%s, CVs were <5.8% within run, <7.5% between runs. No interference was observed from both endogenous compounds, and 33 drugs eventually co-administered during immunosuppression. Over 1,000 patient samples have been analysed by this method in our laboratory in about one year, without any sign of column deterioration.     

Studies on Mercury Pollution: Microdetermination of Mercury in Biological Materials by Cold Vapour Atomic Absorption Spectrometry

Abstract

A simple, rapid, precise and accurate method for the determination of mercury in biological material is described. Biological samples were digested with nitric acid and acidified potassium permanganate and determined by cold vapour analyser. The proposed method was successfully employed for the determination of mercury in samples of fish, hair and blood.     

Solid-Phase Total Synthesis of Cyclosporine Analogues

Syntheses of cyclosporine analogues are reported wherein the peptide couplings were achieved in solid phase. The Wang resin was used as the solid support, and the peptide couplings commenced with the residue 11 of the cyclosporine skeleton. The couplings proceeded in a stepwide manner up to the residue MeBmt¹, using symmetric anhydrides. The peptides were then cleaved off the resin, and the cyclization was achieved in solution using Castro's reagent. The solid-phase synthesis described herein offers a very efficient method for the rapid synthesis of structurally diverse cyclosporine analogues in small quantities. The biological activities of the synthetic cyclosporine analogues were evaluated in two in vitro assays, including the IL-2 reporter gene assay and the cyclophilin binding assay. The structure-activity relationship is discussed.     

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 Paracetamol in Blood Serum Samples by First-Derivative UV Absorption Spectroscopy

Abstract

A rapid method for the determination of paracetamol in human blood serum using first-derivative UV absorption spectroscopy is described. It involves no sample pretreatment, extraction or derivatization procedures, other than a standard deproteinizing technique with trichloroacetic acid. The results can be applied to both therapeutic and toxic levels of paracetamol.     

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.     

The Use of Automated Headspace Gas Chromatography for Determination of 1,1,1-Trichloroethane in Rat Blood and Brain Tissue

Abstract

1,1,1-trichloroethane in blood and brain tissue from rats which had been artificially ventilated with solvent (8000 ppm) was analysed by automated headspace gas chromatography using a fused silica capillary column. A given concentration of 1,1,1-trichloroethane in the brain could be correlated with a corresponding concentration in the blood; both the uptake and release of the solvent were quicker in blood than in brain. No volatile metabolites of the solvent were found. Automated headspace gas chromatographic analysis is a rapid and sensitive technique for the quantitative registration of volatile organic solvents, e.g. of industrial importance, in body fluids and tissues.     

Analysis of Erythromycin II. Determination of Erythromycin in Human Blood Serum by Competitive Displacement of [14C]-Erythromycin from E. Coli Ribosomes

Abstract

A sensitive and selective method for the determination of erythromycin in human blood serum, in the range of 0.1 to 1.0 μg/ml, is described. The procedure is based on extraction of drug into ether and competitive displacement of [¹⁴C]-erythromycin from E. coli ribosomes. The method provides accurate, precise and rapid analyses and should be readily adaptable to bioavailability studies with erythromycin and its salts in man.     

A Sensitive High Performance Liquid Chromatographic Method for U-80, 278A,A Substituted Aminotetralin,in Rat Plasma,Whole Blood and Brain Tissue

Abstract

A sensitive analytical method for U-80, 278A, a substituted aminotetralin analogue in rat plasma, whole blood and brain tissue has been developed. The method involves solid phase extraction, efficient reversed phase HPLC and fluorescence detection, and can measure 1 ng/ml from 50 μl samples. During method development, many analogues were investigated and a wide range of extraction and HPLC conditions were explored. These experiments enabled rapid modification and revalidation of the method to support animal experiments with novel analogues.     

A carbositall electrode modified with a SiO<Subscript>2</Subscript>-hemoglobin-gold film as a promising biosensing element

The analytical prospects of a carbositall electrode modified with a film based on SiO₂, hemoglobin (Hb), and gold nanoparticles (CSE SiO₂-Hb-Au), prepared by deposition with an electrically generated catalyst, are reported. Hemoglobin on the surface of CSE SiO₂-Hb-Au is shown to possess electrocatalytic activity toward dissolved oxygen, giving a rationale for the development of a rapid voltammetric procedure of O₂ quantification with a detection limit of 0.1 mg/L. The inhibition activity of a number of nitrogen-containing organic substances to the catalytic current CSE SiO₂-Hb-Au is studied. A voltammetric procedure is proposed for determination of the anti-flu drug rimantadine, 1-(1-adamantyl)ethylamine hydrochloride, with a detection limit of 0.4 mg/L. The procedure is applicable to the quantification of rimantadine in blood serum and saliva.     

Assessing and Advancing Technology for the Noninvasive Measurement of Clinical Glucose

Abstract

Noninvasive glucose sensing is an active area of research and development with many different approaches being explored since the 1980s. The promise of this approach is to provide a measure of the blood glucose concentration in a rapid, painless, and cost-effective manner so that people with diabetes can better maintain tight glycemic control, thereby reducing the complications of diabetes. This mini-review covers the major approaches to noninvasive glucose sensing and focuses on issues that must be resolved before a clinically useful device can be developed. A strong emphasis is placed on measurement selectivity, and methods to examine and characterize the selectivity of such a complex analytical problem are discussed.     

Thermometric Enthalpy Titration of Proteins

Abstract

A novel, rapid and accurate methodological approach is described for determining the number and type of prototropic groups on proteins. Potentialities of calorimetric titrations are illustrated by their application to the characterization and analysis of a modified protein, viz., ovalbumin diazotized with p-aminobenzoic acid.     

Simultaneous Measurement of Cyclosporine A,Everolimus, Sirolimus and Tacrolimus Concentrations in Human Blood by UPLC–MS/MS

Liquid chromatography coupled with tandem mass spectrometry for therapeutic drug monitoring of immunosuppressants has been widely adopted in clinical chemistry laboratories. However, UPLC is replacing classical LC techniques, providing higher resolution and speed. We developed and validated an UPLC–MS/MS method for the simultaneous measurement of cyclosporine A, everolimus, sirolimus and tacrolimus concentrations in human blood. Following extraction with a zinc sulfate solution and acetonitrile, the chromatographic separation was achieved using an Acquity^® UPLC^® BEH™ (2.1 × 30 mm id, 1.7 µm) reverse-phase C₁₈ column, with a water/methanol linear gradient containing 2 mM ammonium acetate with 0.1 % formic acid at a 0.5 mL min⁻¹ flow rate. All immunosuppressants were detected by ESI mass spectrometry in positive ion multiple reaction monitoring mode using mass-to-charge transitions of 1219.8 → 1202.6/1184.4, 975.5 → 908.3/891.6, 931.5 → 864.3/883.3, 821.4 → 768.2/719.9 for cyclosporine A, everolimus, sirolimus and tacrolimus, respectively. Coefficients of variation and relative bias were less than 5.8 and 9.7 % for cyclosporine A, 8.7 and 6.4 % for everolimus, 8.5 and 7.2 % for sirolimus and 6.7 and 4.7 % for tacrolimus. Limits of quantification were 15.4 µg L⁻¹ for cyclosporine A, 1.42 µg L⁻¹ for everolimus, 1.58 µg L⁻¹ for sirolimus and 0.65 µg L⁻¹ for tacrolimus. Mean recoveries were greater than 77.6 % for all immunosuppressants. Evaluation of the matrix effect showed ion suppression for all the immunosuppressants, except for cyclosporine A, which suffered ion enhancement. No carry-over was observed. The validated method appears to be well adapted for therapeutic drug monitoring of multiple immunosuppressants in daily clinical practice.

     

Biological applications of amide and amino acid containing synthetic macrocycles

ABSTRACT

Amino acid derived macrocycles with elaborate well-defined stereochemistry are a unique class of compounds that have been isolated from natural sources. Macrocycles like cyclosporine, octreotide, and valinomycin have been used in multiple applications, like drugs or ion sensors. Chemists have long been fascinated by the unique molecular recognition capabilities of these macrocycles and tried to design synthetic analogs with similar functions. This article is focused on reviewing current research on amide and amino acid containing macrocycles that have been developed in research laboratories for biological recognition, specifically for anion sensing, ion transport, carbohydrate sensing, and peptide sensing.     

High Performance Liquid Chromatographic Separation and Fluorescent Measurement of Taurine,a Key Amino Acid

Abstract

A rapid, sensitive method for the analysis of taurine in oyster hemolymph (blood) has been developed. Highly fluorescent, substituted isoindoles formed by the reaction of taurine and other amino acids with o-phthaldialdehyde/ethanethiol reagent were separated on a reverse phase, high performance liquid chromatographic column. It was necessary to carefully control the concentration of the sodium ion in the phosphate buffer in order to maintain an adequate resolution of both taurine and tyrosine from β-alanine and arginine. Calibration plots of the fluorescent taurine derivative were linear over 2.5 orders of magnitude with a limit of detection of 0.10 nanomoles per ml of oyster hemolymph.     

Application of Second Derivative Ultraviolet Spectrometry Part II: Determination of Cinnamic Aldehyde and Carvone in Volatile Oils

Abstract

A rapid and simple method for determining cinnamic aldehyde in cinnamon and cassia oils and carvone in caraway and dill oils is presented. These oils are diluted with ethanol and their respective constituents are directly determined by measuring peak-trough amplitudes of the generated second derivative spectra at certain wavelengths. The results obtained are reasonably reproducible with a coefficient of variation less than 2%.     

High-performance liquid chromatographic method for the simultaneous determination of cyclosporine A and its four major metabolites in whole blood

This study aimed to develop a simple and efficient optimized high-performance liquid chromatograph (HPLC) method for simultaneous determination of cyclosporine A (CyA) and its major, partly active metabolites AM1, AM9, AM4N, and AM19 in whole blood from transplant patients using cyclosporine D (CyD) as internal standard. The method used a CN analytical column maintained at 60 °C with hexan-isopropanol (93:7, v/v) as mobile phase; detection was at 212 nm. Linearity for all five compounds was tested in the range of 31-1500 ng ml⁻¹ for CyA and of 31-1000 ng ml⁻¹ for metabolites. The limit of detection was found to be 15 ng ml⁻¹ for all compounds.This modified, inexpensive method is also suitable for measuring cyclosporine A and metabolite concentrations in routine monitoring of patients undergoing treatment with CyA.