Single column high pressure liquid chromatographic determination of drugs in blood.

1. Analysis of anticonvulsants (phenobarbital, diphenylhydantoin and carbamazepine), theophylline and an antiarrhythmic agent (disopyramide) in blood using a simple high pressure liquid chromatography apparatus equipped with a reversed -- phase column is described. A simple extraction of plasma or serum with organic solvent is used to isolate the anticonvulsants and theophylline. Disopyramide is extracted with ether and is further purified by a back extraction into acid. 2. Hexanesulfonic acid -- methanol solutions are used for chromatography of the anticonvulsants and disopyramide while the mobile phase for theophylline is a NH4H2PO4 -- methanol mixture. Chromatographic analysis time for the drugs is approximately 15 minutes. The drugs are monitored by a UV detector at 254 nm except for theophylline which is measured at 280 nm. Quantitation is accomplished by comparison of peak heights with those of internal standards. Quantities of serum or plasma routinely used for analysis are: 200 ul for the anticonvulsants, 100 ul for theophylline and 0.5 ml for disopyramide. Detection limits are less than 1 ug/ml for these quantities.     

Micro-scale method for liquid-chromatographic determination of chloramphenicol in serum.

We describe the use of "high-performance" liquid chromatography to measure chloramphenicol in as little as 25 microL of serum. Serum is treated to precipitate proteins with acetonitrile containing p-nitroacetanilide as an internal standard. Chloramphenicol is eluted with a mobile phase of methanol in pH 7.0 phosphate buffer (35/65 by vol). The drug is measured at 278 nm and simultaneously monitored at 254 nm; interfering substances are detected by examining the 278 nm/254 absorbance ratios. This method is sensitive to less than 0.5 mg/L and the standard curve is linear to at least 50 mg/L. Inter-day precision ranged between 3--6%. We encountered no interference from endogenous compounds or from other drugs we tested.     

Liquid-chromatographic measurement of endogenous and exogenous glucocorticoids in plasma.

The therapeutic response to and side effects of glucocorticoids will be better recognized and the recovery of the adrenals during the tapering of therapy with steroids better evaluated if endogenous and exogenous glucocorticoids are separately assessed. We describe a specific method for simultaneously measuring the concentrations of cortisone, cortisol, prednisone, and prednisolone in plasma by "high-pressure" liquid chromatography. The steroids, together with an internal standard, dexamethasone, are extracted from 1 mL of plasma with methylene chloride-ether, washed with acid and base, and separated isocratically on a normal-phase silica column with a mobile phase consisting of methylene chloride/tetrahydrofuran/methanol/glacial acetic acid (96.85/1/2.1/0.05 by vol) at a flow rate of 1.3 mL/min. The steroids are detected at 254 nm and quantitated by peak-height measurements; their retention times range from 6 to 20 min. The lower limits for routine detection of all four compounds is 10 microgram/L. The analytical recoveries are about 75%; the intra-day variability (CV) is 1 to 9%, and the inter-day variability 2 to 11%. Of 26 drugs and 20 steroids tested, only theophylline presents an interference problem.     

High-performance liquid-chromatographic assay for chloramphenicol in biological fluids

We describe a method for measuring chloramphenicol by high-performance liquid chromatography. The assay involves a single extraction of the biological sample with ether, evaporation of the extract, and chromatography of the residue, redissolved in methanol. A reversed-phase column with an eluting solvent of methanol/water (30/70 by vol) is used. Chloramphenicol is eluted from the column in about 4 min and is well separated from the internal standard (mephenesin), which is eluted at 5.5 min. Absorption of the effluent at 278 nm is monitored and measured. As little as 0.1 microgram of the antibiotic can be analyzed after its extraction from a 0.1-ml sample. The method is suitable for rapid and specific analysis for the drug in plasma, urine, cerebrospinal fluid, and other biological fluids.     

Improved isocratic liquid-chromatographic simultaneous measurement of phenytoin, phenobarbital, primidone, carbamazepine, ethosuximide, and N-desmethylmethsuximide in serum

We describe an improved "high-pressure" liquid-chromatographic assay for simultaneous determination in serum of the five major antiepileptic drugs (ethosuximide, primidone, phenobarbital, phenytoin, and carbamazepine) and N-desmethylmethsuximide (the compound that must be quantitated for therapeutic drug monitoring of the antiepileptic drug methsuximide). Serum protein is precipitated with an acetone solution containing 5-ethyl-5-(p-methylphenyl)barbituric acid as the internal standard. The centrifuged supernate is injected onto the chromatographic column. Drugs and internal standard are eluted at 30 degrees C with mobile phase containing acetonitrile/methanol/phosphate buffer (17/28/55 by vol) at a flow rate of 0.7 mL/min, monitored at 195 nm. Analysis time is about 20 min. Quantitation is by measurement of peak areas. Analytical and absolute recoveries varied from 95 to 104%. Within-day coefficients of variation ranged from 1.6 to 5.4%, between-day CVs from 0.0 to 3.4% in subtherapeutic, therapeutic, and toxic samples. Resolution of therapeutic concentrations of all six drugs was complete. As yet, we have found no drug or drug metabolite that interferes.     

Serum theophylline analysis by high-pressure liquid chromatography.

We have developed and evaluated a rapid, high-pressure liquid-chromatographic method for theophylline in serum. Only 0.2 ml of serum is required for each determination, and the sensitivity of this method is 0.5 mg/liter. This method, involving liquid extraction and silica adsorption chromatography, provides adequate selectivity, accuracy, and precision for routine or research applications. Little sample preparation is required before chromatography. We found no endogenous or exogenous interferences. Use of beta-hydroxypropyl theophylline as the internal standard provides reproducible results for this micro-scale method.     

Liquid-chromatographic separation of urinary 5-hydroxy-3-indoleacetic acid, with measurement at 254 nm

A "high-performance" liquid-chromatographic procedure for 5-hydroxy-3-indoleacetic acid is described and compared with a colorimetric method in which 1-nitroso-2-naphthol is used. The analyte and an internal standard, p-nitrobenzoic acid, were extracted into diethyl ether from urine at pH 4.0 (acidified with HCl) to which sodium chloride had been added, and the ether was back-extracted with acetate buffer, pH 9.2. Aliquots of this extract were injected into a reversed-phase liquid-chromatographic column and eluted with pH 3.5 acetate buffer/methanol (95/5 by vol); the effluent was monitored at 254 nm. The precision (CV) of the method was 11.8% at 1.8 mg/L, 5.5% at 92 mg/L. Analytical recovery averaged 84%. The colorimetric method gave higher values for the analyte than did the chromatographic method for all patients' urines.     

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC ANALYSIS OF THEOPHYLLINE IN SERUM AND ITS USE IN THERAPEUTIC DRUG MONITORING

A rapid, highly sensitive high-performance liquid chromatographic method has been developed for the determination of theophylline in serum using beta-hydroxyethyltheophylline as an internal standard (IS). Theophylline and IS were extracted from serum using a mixture of dichloromethane: isopropanol (90: 10, v/v) and eluted from a 5 microns, C-18 reversed-phase column at 60 degrees C with a mobile phase consisting of sodium acetate buffer-acetonitrile (90:10, v/v), at a flow rate of 1 ml/min with ultraviolet detection at 280 nm. Each analysis required no longer than 7 min to perform. Quantification was achieved by the measurement of the peak: height ratio and the relative and absolute recoveries varied from 86 to 100%. Within-day coefficients of variation ranged from 2.9 to 5.1% and between-day coefficients of variation from 1.1 to 5.9% in subtherapeutic, therapeutic, and toxic concentrations.     

Microanalytical high-performance liquid chromatography assay for cefpirome (HR 810) in serum.

We report a microanalytical high-performance liquid chromatography method for quantitation of cefpirome (HR 810) from serum. The drug was extracted from 0.05 ml of serum with 0.2 ml of isopropanol containing beta-hydroxypropyltheophylline, the internal standard. Separations were performed on a C18 column at ambient temperature with detection at 240 nm. The mobile phase consisted of acetate buffer (0.05 M sodium acetate) containing tetrabutylammonium hydroxide and methanol (pH 5.1; 70:30, vol/vol). The method was linear to 500 micrograms of cefpirome per ml and had a sensitivity of 0.6 micrograms/ml. Analytical recovery was greater than 86%, and the between-day coefficient of variation was less than 4.2%. The stability for 1 week at 4 to 8 degrees C and for 30 days at -20 degrees C was documented. Interference with commonly used antibiotics, analgesics, methylxanthines, and anticonvulsants was not found. The small sample volume and ease of preparation make this method suitable for use in pediatric pharmacokinetic investigations of cefpirome.     

High-performance liquid chromatographic method for determination of cefmetazole in human serum.

A fast, specific, sensitive high-performance liquid chromatographic method for the determination of cefmetazole in human serum was developed. The serum samples were deproteinized by adding 5% trichloroacetic acid in methanol containing barbital as an internal standard and were injected onto a reverse-phase column (mu-Bondapak C18) with a mobile phase of 10 to 15% acetonitrile in 0.005 M citrate buffer (pH 5.4). Eluted components were detected by UV absorption at 254 nm. Cefmetazole and the internal standard were separated from interfering serum components by this method. The peak height ratio of cefmetazole to the internal standard was proportional to the cefmetazole concentration in the range from 0.4 to 100 micrograms/ml. Serum samples obtained from three patients after a single intravenous injection of cefmetazole were assayed by this method and by a microbiological method. There was a good correlation between two assay methods (correlation, coefficient, 0.98). The stability of cefmetazole in human serum was (correlation coefficient, 0.98). The stability of cefmetazole in human serum was also determined by this method. Cefmetazole was stable in human serum for 2 weeks at 4 degrees C or for at least 8 weeks if it was kept frozen. As the high-performance liquid chromatography method is simple, specific, accurate, and reproducible, it appears to be more suitable for routine assay of cephalosporins than other assay methods.     

Liquid-chromatographic determination of cephalosporins and chloramphenicol in serum

A "high-performance" liquid-chromatographic technique involving a radial compression module is used for measuring chloramphenicol and five cephalosporin antibiotics: cefotaxime, cefoxitin, cephapirin, and cefamandol. Serum proteins are precipitated with acetonitrile solution containing 4'-nitroacetanilide as the internal standard. The drugs are eluted with a mobile phase of methanol/acetate buffer (30/70 by vol), pH 5.5. Absorbance of the cephalosporins is monitored at 254 nm. Standard curves are linear to at least 100 mg/L. The absorbance of chloramphenicol is monitored at 254 nm and 280 nm, and its standard curve is linear to at least 50 mg/L. The elution times for various other drugs were also determined, to check for potential interferents.     

High performance liquid chromatographic procedure for the simultaneous determination of theophylline, caffeine, and phenobarbital in neonates

A sensitive HPLC method is reported for the simultaneous determination of theophylline, caffeine, and phenobarbital in 100 microliters of plasma. After a single extraction of the drugs with chloroform/isopropanol (90 + 10 by volume) at low pH in the presence of an excess of ammonium sulphate they are resolved and quantified using a reversed-phase column (Spherisorb 5 ODS). The drugs are eluted with a binary-solvent gradient system (acetonitrile/phosphate buffer pH 4.6) at room temperature and monitored at 204 nm. Quantitation is based on peak-height ratio of analyte to interval standard (8-chlorotheophylline). Complete chromatographic resolution of all drugs is achieved within 15 min. The method is linear to 40 mg/l of theophylline and caffeine, and to 80 mg/l of phenobarbital. Numerous drugs and xanthine metabolites tested do not interfere.     

High-performance liquid chromatographic determination of floctafenine and its major metabolite, floctafenic acid in plasma.

A simple, rapid and selective high-performance liquid chromatographic method for the determination of floctafenine and its major metabolite, floctafenic acid, in plasma is reported. Plasma samples were purified using the mobile phase as a protein precipitant. The supernatant containing parent compounds and diazepam (internal standard) were eluted from a 5 micron C 18 reversed-phase column at ambient temperature. The mobile phase consisted of 0.05 M sodium acetate:acetonitrile: methanol (200:100:100 v/v/v) adjusted to pH 5 and pumped isocratically at a flow rate of 1.5 ml/min. The effluent was monitored at 350 nm. Quantification was achieved by the measurement of the peak-height ratio of each drug to the internal standard. The mean percentage recoveries from plasma samples spiked with floctafenine and floctafenic acid ranged from 88.13 to 101.93%. Detection limits were 100 ng/ml for floctafenine and 50 ng/ml for floctafenic acid. The coefficients of variation (RSD, %) for within-day and day-to-day analysis were less than 8%.     

Liquid-chromatographic determination of five orally active cephalosporins--cefixime, cefaclor, cefadroxil, cephalexin, and cephradine--in human serum

We report an isocratic "high-performance" liquid-chromatographic (HPLC) procedure for measurement of five orally administered cephalosporins (cefixime, cefaclor, cefadroxil, cephalexin, and cephradine) in 0.1 mL of human serum. Serum protein is precipitated with acetonitrile, the sample is centrifuged, and the supernate is evaporated under nitrogen. The residue is reconstituted in 0.1 mL of mobile phase, and 50 to 80 microL of this is injected onto a reversed-phase Altex Ultrasphere Octyl (C8) column. The five cephalosporins are resolved by elution with a pH 2.6 mobile phase of methanol/monobasic phosphate buffer (20/80) by vol), flow rate 2 mL/min. The column effluent is monitored at 240 nm. Cefixime serves as the internal standard for the analysis of the four other compounds, cephalexin as the internal standard for cefixime. We used two standard curves for all compounds: a low-range curve for concentrations commonly observed clinically and a higher-range curve for higher concentrations. The former were linear from 1.0 to 10 mg/L for cefaclor, cefadroxil, cephalexin, and cephradine and from 0.1 to 1 mg/L for cefixime. The high-concentration curves were linear from 1 to 10 mg/L for cefixime and from 10 to 100 mg/L for the other compounds. The detection limits were 0.1 mg/L for cefixime, 1 mg/L for the other cephalosporins. Mean within-run and day-to-day CVs were always less than 15% for all compounds studied.     

Concurrent measurement of flecainide acetate and propranolol by normal-phase high-performance liquid chromatography

This simple, isocratic, normal-phase liquid-chromatographic method concurrently measures flecainide acetate and propranolol in 100 microL of serum within 8 min. The chromatographic system consists of a Waters "Resolve" column packed with 5-microns silica spheres and a mobile phase of ammonium sulfate (10 mmol/L, pH 6.8)/methanol (22/78 by vol), pumped at 0.9 mL/min and monitored by a fluorometer (excitation at 225 nm and emission at 340 nm). After 100 microL of serum is mixed with 200 microL of the internal standard solution [N-(2-piperidylmethyl)-2,3-bis(2,2,2-trifluoroethoxy)-benzamide HCl, 2500 micrograms/L] and 200 microL of 0.2 mol/L sodium carbonate, the sample is extracted into butanol/hexane (20/80 by vol). The organic layer is separated and evaporated, and the residue is redissolved in 200 microL of methanol; 50 microL of this is injected onto the column. Relative recovery was 100% over the assay range of 25-2000 micrograms/L for flecainide and 10-2000 micrograms/L for propranolol. Within-run CVs were less than 2% for flecainide and less than 5% for propranolol; day-to-day CVs ranged from 5.0% to 6.5% for flecainide and from 3% to 12% for propranolol.     

Liquid-chromatographic determination of alpha- and gamma-tocopherols in erythrocytes, with fluorescence detection

We have adapted to erythrocytes a method for the determination of alpha- and gamma-tocopherols in plasma and platelets. Erythrocytes (50 microL) were extracted with methanol containing tocol (internal standard) and pyrogallol. Tocopherols were partitioned into chloroform, washed, and injected in methanol onto a reversed-phase (C18) "high-performance" liquid-chromatographic column. The mobile phase was methanol/water (99/1 by vol) at a flow rate of 2 mL/min and detection was with a "high-performance" spectrophotofluorometer. The limit of detection for either tocopherol is 0.10 microgram/mL of packed cells. Analytical recoveries ranged from 93 to 104%. Some values for tocopherols in human erythrocytes are presented.     

Simultaneous measurement of ethosuximide, primidone, phenobarbital, phenytoin, carbamazepine, and their bioactive metabolites by liquid chromatography

A simple, isocratic liquid-chromatographic method was developed for simultaneously measuring ethosuximide, primidone, phenobarbital, phenytoin, carbamazepine, and their bioactive metabolites within 10 min. The chromatographic system involves a Waters' Radial-NOVA PAK C18 reversed-phase column and acetone/methanol/acetonitrile/10 mmol/L phosphate buffer (10/21/8/61 by vol, pH adjusted to 7.95 with NaOH) as mobile phase. The antiepileptic drugs are extracted from 50 microL of serum by mixing with 50 microL of acetonitrile containing 10 mg of tolybarb per liter as internal standard. After centrifugation, 20 microL of the supernate is injected onto the column and eluted with mobile phase at the rate of 2.8 mL/min at ambient temperature. The column effluent is monitored at 200 nm. The method can detect the five antiepileptic drugs in concentrations as low as 0.5 mg/L. Analytical recovery ranges from 98 to 102%. Within-run CV ranged from 2.9 to 5.8% and between-run CV from 4.7 to 7.1%. The method can also be used to measure N-desmethyl-methsuximide, chloramphenicol, and pentobarbital.     

HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC ANALYSIS OF CAFFEINE CONCENTRATIONS IN PLASMA AND SALIVA

A rapid high performance liquid chromatographic (HPLC) method for the analysis of caffeine in plasma and saliva is described. Samples of saliva and plasma were purified using zinc sulphate solution as protein precipitant. The supernatant was injected directly onto the column. The mobile phase consisted of ammonium acetate buffer:acetonitrile:methanol (82:15:3, v/v). Measurements were carried out at 254 nm. Acetanilide was used as the internal standard and analysis was completed in 10 min. No interference from endogenous components or other methylxanthines was observed. The coefficients of variation for within day and between day analysis for both saliva and plasma were less than 7.66%. Samples were collected from 20 volunteers. The correlation coefficient between plasma and saliva caffeine concentrations was found to be 0.98.     

Simultaneous quantification of sirolimus, everolimus, tacrolimus and cyclosporine by liquid chromatography-mass spectrometry (LC-MS).

We developed a universal liquid chromatography-mass spectrometry (LC-MS) assay with automated online extraction to quantify simultaneously the immunosuppressants sirolimus, everolimus, tacrolimus, and cyclosporine. Whole blood (300 microl) plus 6 microl 32-desmethoxyrapamycin (1 ng/microl) as internal standard was treated with 600 microl methanol/0.2 M ZnSO4 (80/20 v/v). After vortexing (30 s) and centrifugation (20000 g, 5 min) 50 microl of the supernatant were loaded on an extraction column, were washed by 0.35 ml/min water for 3 min and, after activation of a column-switching valve, were back-flushed by 0.25 ml/min methanol/ water (90/10 v/v) onto a C18 analytical column. After 22 min the extraction column was washed for 2 min with methanol and for 3 min with water before starting the next run. Column temperatures were kept at 33 degrees C. Sodium adduct ions [M+Na]+ ions were detected in the selected ion mode. For sirolimus, everolimus and tacrolimus the assay was linear from 0.3 to 200 microg/l and for cyclosporine from 5 to 1000 microg/l (all r2>0.999). Recovery of all immunosuppressants and the internal standard was >90% and in general, inter-day and intraday precision was <10%. The simultaneous quantification of blood levels by LC-MS seems to be the method of choice in transplanted patients receiving multiple immunosuppressants.     

High–performance liquid chromatographic determination of perphenazine in plasma

A reversed–phase high–performance liquid chromatographic (HPLC) method for the analysis of perphenazine in rabbit plasma was developed. Amitriptyline HC1 was used as the internal standard. The chromatography was performed using a Microsorb–CN column; the mobile phase consisted of 85:15 methanol/0.005 M ammonium acetate buffer; flow rate of 1.6 ml/min; and ultraviolet detection at 254 nm. Retention times were 10.8 and 18.1 min for perphenazine and amitriptyline HC1, respectively. The recovery of perphenazine added to plasma in the concentration range of 0.055 μg/ml was found to be greater than 80%. This procedure was used for the analysis of plasma samples collected over time following the oral administration of perphenazine tablets in rabbits.