Simple and simultaneous determination for 12 phenothiazines in human serum by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed for the simultaneous analysis of the 12 phenothiazines (chlorpromazine, fluphenazine, levomepromazine, perazine, perphenazine, prochlorperazine, profenamine, promethazine, propericiazine, thioproperazine, thioridazine and trifluoperazine) in human serum using HPLC/UV. The separation was achieved using a C(18) reversed-phase column (250 mm x 4.6 mm I.D., particle size 5 microm, Inersil ODS-SP). The mobile phase, consisting of acetonitrile-methanol-30 mM NaH(2)PO(4) (pH 5.6) (300:200:500, v/v/v), was delivered at a flow rate of 0.9 mL/min and UV detection was carried out at 250 nm. The recoveries of the 12 phenothiazines spiked into serum samples were 87.6-99.8%. Regression equations for the 12 phenothiazines showed excellent linearity, with detection limits of 3.2-5.5 ng/mL for serum. The inter-day and intra-day coefficients of variation for serum samples were commonly below 8.8%. The selectivity, accuracy and precision of this method are satisfactory for clinical and forensic purposes. This sensitive and selective method offers the opportunity for simultaneous screening and quantification of almost all phenothiazines available in Japan for the purposes of clinical and forensic applications.     

A reversed-phase high-performance liquid chromatographic method for the determination of aloesin, aloeresin A and anthraquinone in Aloe ferox

A reversed-phase HPLC method for the quantification of aloesin, aloeresin a and anthraquinone (as barbaloin) in Aloe ferox Miller and aloe-related products has been developed and validated. The method utilized a C18 column with a water-methanol gradient and UV detection at 297 nm. The method validation included linearity, accuracy, precision, limit of detection, limit of quantitation, specificity and standard solution stability. The method showed good linearity (r > 0.99 for all components) and recovery (>85% for all components). The detection and quantitation limits for barbaloin were determined to be 0.02 and 0.1 ppm at signal-to-noise ratios of approximately 3:1 and 10:1, respectively.     

A reversed-phase high-performance liquid chromatographic method for characterization of biosynthetic human growth hormone

An isocratic reversed-phase high-performance liquid chromatographic method for the determination of human growth hormone (HGH) purity is described. This method offers superior resolution of HGH-related substances (e.g., sulfoxide and desamido derivatives) from unmodified HGH when compared to a number of alternative chromatographic and electrophoretic techniques.     

Rapid high-performance liquid chromatographic determination of serum gabapentin

Gabapentin (GBP) is a new antiepileptic drug approved for clinical treatment of partial seizures in the USA. Serum GBP concentrations in 283 patients were studied using high-performance liquid chromatography with fluorescence detection. The standard curves were linear over a range of 60 ng to 15 μg/ml. The coefficient of variations were 3.4 to 8.8% and 1.4 to 9.8% for intra- and inter-assay studies, respectively. The lower limit of quantitation was 10 ng/ml. Of the 283 patients studied, 72.5% had GBP levels between 2 and 10 μg/ml, 14.8% were below 2 μg/ml and 12.7% above 10 μg/ml. The mean±S.E. of GBP in 283 patients was 5.38±0.23 μg/ml. Peak concentrations of more than 15 μg/ml and trough levels as low as 0.1 μg/ml were not uncommon. The method described was rapid, simple, highly sensitive and reproducible. Other antiepileptic drugs and endogenous compounds did not interfere with the assay.     

A high-performance liquid chromatographic method for the determination of polyphosphoinositides in brain

A simple high-performance liquid chromatographic method for the determination of endogenous phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) in brain has been developed. PIP and PIP2 were derivatized with 9-anthryldiazomethane to yield (9-anthryl)PIP and di(9-anthryl)-PIP2. The derivatives were separated on a reversed-phase column using isocratic elution and detected with a uv detector. The detection limits of PIP and PIP2 were 0.25 micrograms. The method with uv detection was sufficiently sensitive to measure the concentrations of PIP and PIP2 in rat brain. The levels of PIP and PIP2 were increased in developing rat brain and were decreased after 10 min of ischemia.     

A new high-performance liquid chromatographic method for determination of warfarin enantiomers

Warfarin is the most common agent used for control and prevention of venous as well as arterial thromboembolism. Although warfarin is administered as a racemic mixture of two stereoisomers (S and R), the S-form is mainly responsible for the anticoagulant effect. The anticoagulant effect of the drug is monitored by analysis of prothrombin complex (International Normalised Ratio,INR). In some cases, however, the measurements of plasma warfarin concentration are needed. Here, we present a new, rapid, sensitive and cost-effective HPLC-method for the determination of warfarin enantiomers in plasma. The chromatographic system consisted of Waters 616 gradient pump, Waters 996 photo diode array detector, Gilson 230 autoinjector and Pirkle (R,R) Whelk-O1 column (25 cmx4.6 mm I.D., 5 microm). An isocratic mobile phase of methanol/acetonitrile/water (50/10/40, v/v) with 0.1% glacial acetic acid was used. The follow rate was 1 mL/min. Data analysis was carried out with Waters Millennium32. The absorbance at 305 nm was measured with a total run-time of 15 min. Method linearity was studied by establishing regression data containing eight points over the range 0.08-10 microg/mL. In this range, warfarin showed to be linear (r2=0.9997 for S-warfarin and r2=0.9998 for R-warfarin). The limit of detection in plasma was 16 ng/mL for S-warfarin and 18 ng/mL for R-warfarin. Limit of quatitation was defined as 10xLOD. The extraction recovery was approximately 80%. Also the relation between INR and warfarin concentration was investigated. As expected, there was a low correlation between these two variables (r=0.23, y=0.3044x+0.9712). This method offers a rapid and cost-effective determination of warfarin enantiomers in human plasma.     

A high-performance liquid chromatographic method for the determination of dolichyl phosphates in tissues

A sensitive high-performance liquid chromatographic method has been developed for the determination of dolichyl phosphate homologs in tissues. Dolichyl phosphates were methylated and derivatized with 3-(9-anthryl)-diazo-2-propene to yield methyl 3-(9-anthryl)-2-propenyl dolichyl phosphates. As an internal standard, 2,2-didecaprenylethyl phosphate was used. The derivatives were separated on a reversed-phase column using isocratic elution, and detected fluorometrically. The method with fluorescence detection was sufficiently sensitive to measure the concentration of dolichyl phosphate homologs in tissues.     

Capillary reversed-phase high-performance liquid chromatographic determination of acetyl-methylprednisolone in feline spinal cord

Capillary reversed-phase high-performance liquid chromatography (RP-HPLC) was used to determine acetylmethylprednisolone (A-MP) that had been administered to feline spinal cord tissue. The method used a 300 mm × 0.32 mm I.D. packed capillary octadecylsilyl (ODS) column and an isocratic mobile phase of 40 mM triethylamine formate (TEAF, pH 3.2)-acetonitrile (50:50, v:v). The chromatographic behavior of A-MP was evaluated with respect to peak-area and peak-height by varying the A-MP concentration (12–190 μM) with a fixed injection volume (1 μl), and by varying the injection volume (1–10 μl) with a fixed concentration (12 μM) of A-MP. The limit of detection (signal-to-noise ratio, 3:1) was 250 pg (600 fmol) of synthetic A-MP. Various amounts of A-MP directly spiked into feline spinal cord segments were solvent extracted, separated, and plotted against peak-area (r² = 1.00). Background tissue without A-MP gives minimal (<1%) interference at 243 nm. The method also detects exogenous A-MP that was administered into feline spinal cord via an intrathecal injection. Furthermore, the presence of A-MP was confirmed via its molecular ion and corresponding product ions that were obtained by fast-atom bombardment tandem mass spectrometry (FAB-MS-MS).     

Simultaneous reversed-phase high-performance liquid chromatographic separation of non-polar isoprenoid lipids and their determination

A procedure for the rapid identification and determination of non-polar isoprenoid lipids from animal tissues was developed. The complete determination can be carried out by reversed-phase HPLC of just two samples. The first, extracted from unaltered tissues and suitably processed by column chromatography, provides information about free cholesterol, cholesteryl esters, coenzymes Q, free dolichols and dolichyl esters. The second, obtained from saponified tissues, can be used to detect both total cholesterol and total dolichols. Specific calibration graphs were constructed for the determination of the different constituents.     

Solid-phase extraction and reversed-phase high-performance liquid chromatographic determination of sulphur mustard in blood

A reversed-phase high-performance liquid chromatographic method is reported for the analysis of sulphur mustard in blood with the aid of solid-phase extraction sample preparation. Sulphur mustard is extracted from blood samples (both in vitro and in vivo) of rats with a solution of 0.05 M sodium dodecyl sulphate and pre-concentrated over Sep-Pak C₁₈cartridges pre-coated with Tween-20. A Polygosil C₁₈ column is used with acetonitrile—water (52:48, v/v) as mobile phase for separation and sulphur mustard was detected at 200 nm.     

Improved high-performance liquid chromatographic assay for the determination of ethionamide in serum

A solid-phase extraction (SPE) method was developed to simplify the preparation of human serum prior to high-performance liquid chromatography of ethionamide (ETA). Octadecyl SPE columns were used. Serum constituents were removed from the column with water, and ETA was eluted with methanol. Samples were evaporated to dryness, reconstituted in mobile phase, and assayed. The method is reproducible, with a recovery of ETA of 64%, comparable to the more tedious liquid-liquid extraction method for ETA.     

High-performance liquid chromatographic method for the simultaneous determination of monoethylglycinexylidide and lignocaine

An accurate and sensitive high-performance liquid chromatographic method with UV detection was developed for the simultaneous measurement of monoethylglycinexylidide (MEGX) and lignocaine in human plasma and serum, using organic solvent extraction and trimethoprim (TMP) as an internal standard. The mean recoveries for MEGX, TMP and lignocaine were 86.1 ± 3.7, 98.3 ± 1.8 and 77.0 ± 4.7%, respectively (n = 6). The relative standard deviations for MEGX concentrations of 10 and 200 ng/ml were < 4% and for lignocaine concentrations of 200 and 1200 ng/ml they were < 8%.     

Simple and robust high-performance liquid chromatographic method for the determination of ranitidine in microvolumes of human serum

A simple robust high-performance liquid chromatographic method is described for the determination of ranitidine in microvolumes of human serum. The drug of interest was isolated using liquid–liquid extraction with dichloromethane and back-extraction with 0.1% phosphoric acid and separation was obtained using a reversed-phase column under isocratic conditions, with ultraviolet detection at 313 nm. Intra-day and inter-day coefficients of variation ranged from 1 to 6% and 3 to 10%, respectively. Accuracy of the assay was less than 10% at all concentrations. The limit of detection and the limit of quantitation were 2 and 7 ng/ml, respectively. The linearity was assessed in the range 10–1000 ng/ml. It was shown that a group of common drugs co-administered with ranitidine did not interfere with its determination. The applicability of this method for the pharmacokinetic study of ranitidine following i.v. infusion in patients was demonstrated using only 100 μl of serum. The ruggedness of the assay was demonstrated over a three-year period.     

A method for determination of iododeoxyuridine substitution of thymidine using reversed-phase high-performance liquid chromatography

An assay for thymidine substitution by iododeoxyuridine (IdUrd) using reversed-phase high-performance liquid chromatography (HPLC) has been developed. Three principal steps in this procedure are: extraction of DNA from cell or tissues, hydrolysis of DNA into deoxynucleosides and separation using HPLC. Approximately 1 microgram of DNA was recovered from 10(5) cells by phenol extraction, and subjected to hydrolysis into deoxynucleosides which required a three-stage DNA digestion using enzymes DNAse I. phosphodiesterase I and alkaline phosphatase. The deoxynucleosides were separated on the Microsorb C18 column with isocratic elution; 90-100% of the DNA was recovered as deoxynucleosides on the column. The method was used to determine quantitatively the percent IdUrd substitution of thymidine in Chinese hamster lung cells in vitro and BA1112 rhabdomyosarcoma in WAG/Rij rats perfused with IdUrd. It was possible to determine the thymidine substitution by IdUrd as small as 1% using a few micrograms of DNA. The close correspondence between the percent substitutions determined by HPLC and those determined by radioactive assay using [125I]-labelled IdUrd, confirmed the accuracy of our HPLC method. The HPLC analysis is especially suitable for the determination of percent IdUrd substitution of thymidine in tissue biopsies from animals used in in vivo experiments or humans undergoing radiation treatment.     

Rapid high-performance liquid chromatographic method for the determination of ketamine and its metabolite dehydronorketamine in equine serum

A simple, rapid and sensitive high-performance liquid chromatographic procedure has been developed for the determination of ketamine and dehydronorketamine in equine serum. Sample preparation consisted of mixing equal volumes of serum and acetonitrile—phosphoric acid (85%)—water (20:2:78, v/v/v), followed by ultrafiltration through a 10 000 molecular mass cut-off filter. Separation of these two analytes in the ultrafiltrate was accomplished on a reversed-phase phenyl column eluted with methanol—acetonitrile—phosphate buffer solution. Ketamine and dehydronorketamine were detected by a variable photometric UV-Vis detector set at 215 nm, and confirmed by a photodiode array detector operated in the 200–320 nm range. The limit of detection for ketamine was 5–15 ng/ml in equine serum. Additionally, the dehydronorketamine peak identity was tentatively confirmed by thermospray liquid chromatography—mass spectrometry.     

Reliable and specific high-performance liquid chromatographic method for simultaneous determination of loratadine and its metabolite in human plasma

A high-performance liquid chromatographic (HPLC) method with fluorescence detection has been developed for the simultaneous determination of loratadine (L) and its metabolite, descarboethoxyloratadine (DCL), in human plasma. Following a two-step liquid-liquid extraction with toluene, the analytes were separated using a gradient mobile phase consisting of methanol-acetonitrile-phosphate buffer. The linearity for L and DCL was within the concentration range of 0.5-16 ng/ml. The coefficient of variation of intra- and inter-day assay was <8.3%, with accuracy ranging from 98.3 to 105.7%. The lower limit of quantification was 0.5 ng/ml for both L and DCL. This method has been demonstrated to be reliable, and is an improvement over existing methods due to its capability for determining L and DCL simultaneously in a single chromatographic run.     

A high-performance liquid chromatographic method for the determination of pseudouridine and uric acid in native human urine and ultrafiltrated serum

A simple and reliable HPLC method for quantitative determination of pseudouridine and uric acid in human urine and serum using a cation-exchange resin is described. This method is straightforward (12 runs of urine samples per day since the sample is only diluted into buffer and then chromatographed), sensitive, and highly reproducible. The column is stable over long periods (3 months of uninterrupted use at a time; it is thereafter easily restored to the original state). Mean excretion values for pseudouridine (in μmol/mmol creatinine) are 26.4 ± 3.1 (17 female adults), 23.8 ± 2.5 (12 male adults), 164.7 ± 32.2 (37 male preterm infants); mean values for uric acid (μmol/mmol creatinine) are, respectively, 310.3 ± 90.5, 278.2 ± 56.1, and 1108 ± 314. Human serum is deproteinized by pressure ultrafiltration in microcollodion bags with a nominal exclusion molecular weight of 12,400 and then put directly onto the HPLC column. The complete procedure takes 4 h.