Pokar M. Kabra

Solid-phase extraction and determination of dansyl derivatives of unconjugated and acetylated polyamines by reversed-phase liquid chromatography: improved separation systems for polyamines in cerebrospinal fluid, urine and tissue

A sensitive and simple liquid chromatographic assay with fluorometric detection for unconjugated and acetylated polyamines in biological fluids is described. After precolumn derivatization with dansyl chloride, unconjugated polyamines and acetylated polyamines were extracted by elution from a Bond-Elut C18 column and then separated on a reversed-phase column with gradient elution. The complete analysis of unconjugated putrescine, spermidine, and spermine in either hydrolyzed urine, cerebrospinal fluid or tissue could be accomplished within 20-26 min, while the simultaneous analysis of unconjugated polyamines and monoacetylpolyamines could be completed within 40 min. Unhydrolyzed urine and cerebrospinal fluid required a Bond-Elut cation-exchange clean-up before dansylation. Standard curves for the assay were linear up to 20 nmol/ml, and the within-day and day-to-day coefficients of variation were between 1.1 and 4.6% and between 1.6 and 11.8%, respectively. Results obtained with the method were compared with results obtained with a well established modified amino acid analyzer method for urine, tissue and cerebrospinal fluid samples. The correlation coefficients between these two methods were in the range 0.933-0.996. Detection limits between 50 and 150 fmol were achieved for unconjugated and acetylated polyamines. Of more than twenty drugs and amines tested for possible interference with the assay, only normetanephrine was found to have the same retention time as the internal standard 1,6-diaminohexane.

High-pressure liquid chromatographic analysis of diazepam, oxazepam and n-desmethyldiazepam in human blood

We describe a rapid method for precisely measuring concentrations of diazepam, oxazepam and N-desmethyldiazepam in blood by high-pressure liquid chromatography. The drugs, together with an internal standard, prazepam, are extracted from 2 ml of blood and analyzed isocratically on a reversed-phase column with a mobile phase consisting of acetonitrile-0.01 M sodium acetate buffer (35:65 v/v). The eluted drugs are detected by their absorption at 240 nm. The sensitivity of this method is 30 microgram/l for oxazepam and N-desmethyldiazepam, 40 microgram/l for diazapam, for 2-ml blood samples. Relative recovery of added drugs to blood varies from 91 to 110%. The day-to-day precision (coefficient of variation) established by 10 replicate analyses was 2.8 to 9.6%.

Simultaneous liquid Chromatographie analysis of amitriptyline, nortriptyline, imipramine, desipramine, doxepin, and nordoxepin

A simultaneous method for the therapeutic monitoring of amitriptyline, doxepin, imipramine, and their active demethylated metabolites nortriptyline, nordoxepin, and desipramine, respectively, in plasma or serum by reversed-phase liquid chromatography (RPLC) is presented. The drugs and the internal standard (loxapine) are first extracted from 2 ml of serum into butylchloride at pH 14, and then back extracted into 200 microliter of 0.025 mol/l hydrochloric acid. An aliquot of the aqueous acid phase is injected into the chromatograph and eluted with acetonitrile-phosphate buffer (21: 79, by vol.) containing 0.6 nl of n-nonylamine per liter of phosphate buffer. The drugs are eluted in a total chromatographic time of approximately 13 min at ambient temperature and detected at 200 nm. A sensitivity of 5 microgram/l of serum for each drug is obtained. Recoveries for these drugs ranged from 77% to 103%; and the coefficient of variation (day-to-day) ranged from 4.2 to 7.8. Of 35 basic or neutral drugs tested for possible interference, only propoxyphene interferes with the analysis of nortriptyline.

Improved solid-phase extraction and liquid chromatography with electrochemical detection of urinary catecholamines and 5-S-l-cysteinyl-l-dopa

We describe a rapid, precise, accurate liquid chromatographic procedure for determining urinary catecholamines and 5-S-L-cysteinyl-L-dopa. The catecholamines (norepinephrine, epinephrine, and dopamine) and 5-S-L-cysteinyl-L-dopa are extracted from 1.0 ml of urine together with internal standards, by using a Bond-Elut strong cation-exchange (SCX) and an affinity phenylboronic acid (PBA) extraction column in series. The eluate obtained from PBA column is then chromatographed on a reversed-phase C18 column with a mobile phase containing pentane- and heptanesulfonate as ion-pair reagents. The detection is achieved with an amperometric detector set at an oxidation potential of +0.55 V. The chromatography is complete is less than 8 min for catecholamines and less than 5 min for cysteinyldopa. The method can measure less than 2 micrograms/l for catecholamines and 5 micrograms/l for cysteinyldopa. Analytical recoveries of catecholamines and cysteinyldopa added to urine pool ranged from 90-107%. Between run coefficient of variation ranged from 4.7 to 8%. None of the drugs and catecholamines metabolites tested interfered with the assay.

Liquid-Chromatographic Determination of Antidysrhythmic Drugs: Procainamide, Lidocaine, Quinidine, Disopyramide, and Propranolol

Summary We present a method for the analysis of antidysrhythmic drugs [procainamide, acecainide (NAPA2), lidocaine, quinidine, disopyramide, N-desisopropyl disopyramide, and propranolol] in serum. The drugs, together with an internal standard, are extracted from 0.2–1.0 ml of serum, separated on an octyl-bonded reversed-phase column using a mobile phase consisting of acetonitrile/phosphate buffer, and monitored by either ultraviolet or fluorescence spectrophotometry. The proposed method offers good reproducibility, sensitivity, linearity, and accuracy. Of more than 50 drugs and metabolites tested for possible interference, only diazepam, flurazepam, and the N-oxide metabolite of quinidine interfere with quinidine analysis, while meperidine coelutes with disopyramide. However, diazepam and flurazepam do not interfere with quinidine analysis with fluorescence detection.

Liquid chromatography in clinical analysis

To Liquid Chromatography.- Principles of Liquid Chromatography.- Instrumentation for Liquid Chromatography.- Liquid Chromatography Column Technology.- Therapeutic Drug Monitoring and Toxicology.- Why Measure Drug Levels?.- Anticonvulsants.- Theophylline and Antiarrhythmics.- Antibiotics.- Tricyclic Antidepressants.- Antineoplastic Drugs.- Hypnotics and Sedatives.- Toxicology Screening.- Clinical Analysis of Endogenous Human Biochemicals.- Determination of Tyrosine and Tryptophan Metabolites in Body Ruids Using Electrochemical Detection.- Steroids.- Proteins.- Bilirubin and Its Carbohydrate Conjugates.- Porphyrins.- Organic Acids by Ion Chromatography.- Major and Modified Nucleosides, RNA, and DNA.- Polyamines.

Liquid chromatographic analysis of clonazepam in human serum with solid-phase (Bond-Elut®) extraction

A simple, sensitive, selective and precise liquid-column chromatographic assay for clonazepam is described, in which 1 ml of serum containing 50 micrograms/l methylclonazepam as an internal standard is extracted by elution from a Bond-Elut column with 400 microliter of methanol. An aliquot of the eluate is injected on to a reversed-phase column and eluted with a mobile phase of acetonitrile--phosphate buffer (30:70) at a flow-rate of 2 ml/min at a column temperature of 50 degrees C. Detection is at 254 nm. Chromatography is complete in 12 min. A sensitivity of 2 ng/ml is attained when 1 ml of serum is extracted. Analytical recovery of the clonazepam added to serum ranged from 91% to 99% with a coefficient of variation of 6.0%. This assay for clonazepam has good precision, with coefficients of variation of 11% at 15 ng/ml and 2.6% at 50 ng/ml. There was no interference from any of the commonly used antiepileptics.

Clinical analysis of individual steroids by column liquid chromatography.

At present, there are various LC methods available for the determination of estriol, aldosterone, and cortisol in urine, and for cortisol, cortisone, 11-deoxycortisol, corticosterone, 17-hydroxyprogesterone, estriol, vitamin D isomers, and various exogenous glucocorticoids in serum. The LC methods are more specific than the currently available RIAs or homogenous immunoassays. However, whether the data obtained by more specific LC methods are better clinically than the commonly used immunoassays for these steroids must still be proven. In this review, I have critically evaluated various LC methods currently available for the routine determination of clinically important steroids in the clinical laboratories. A complete evaluation and advantages and disadvantages of alternative techniques are beyond the scope of this review. However, the readers are urged to refer to the review articles and chapters listed in the reference section.

Liquid chromatography with fluorometric detection of unconjugated estriol in serum of pregnant women.

We describe a fluorometric liquid chromatographic assay for the measurement of unconjugated estriol in the serum of pregnant women. Estriol is extracted into methylene chloride/propanol-2 from serum by use of a Clin-Elut extraction column, the extract evaporated, and the residue redissolved in mobile phase. An aliquot is injected onto the liquid chromatograph and the estriol is separated on a reversed-phase octyl column with a mobile phase consisting of acetonitrile/phosphate buffer (23:77, v/v). The effluent is monitored by fluorescence detection. The proposed method offers good reproducibility (CV less than 7%), sensitivity (less than 0.5 micrograms/l), and accuracy. Of many drugs and steroids tested, only 16,17-epiestriol interferes with the estriol analysis.

Liquid chromatographic determination of cyclosporine in whole blood with the advanced automated sample processing unit

We describe a rapid, precise, cost-effective, and accurate isocratic liquid chromatographic (LC) procedure for determining cyclosporine in whole blood. The cyclosporine is extracted from 0.5 ml of whole blood together with 200 micrograms of cyclosporin D, added per liter as internal standard, by using an Advanced Automated Sample Processing (AASP) unit. The on-line solid-phase extraction is performed on an octasilane sorbent cartridge which is interfaced with a Perkin-Elmer 83 X 4.6 mm I.D. cartridge column, packed with 3-micron octadecyl packing. The column is eluted with a mobile phase containing acetonitrile-water (13:7) at a flow-rate of 1.0 ml/min at a column temperature of 70 degrees C. The column effluent is monitored at 210 nm. The absolute recovery of cyclosporine exceeded 87% and the linearity extended up to 2000 micrograms/l. Within-run and day-to-day coefficients of variation were less than 8%. The correlation between AASP-LC and manual Bond-Elut extraction-LC method was excellent (r = 0.97).