A new on-line, in-tube pre-column derivatization technique for high performance liquid chromatographic determination of azithromycin in human serum

Pre-column derivatization methods for high performance liquid chromatographic assay of specific pharmaceutical agents using 9-fluorenylmethyl chloroformate (FMOC-Cl) have received special attention because highly fluorescent and stable adducts are provided by these methods. However, unlike the post-column on-line techniques, long derivatization time is needed and the reaction cannot be well controlled. A new, sensitive and fast pre-column on-line derivatization technique coupled with high-performance liquid chromatography using FMOC-Cl as labeling agent is described and validated for determination of azithromycin in human serum. After extraction of the drug from serum, the residue was reconstituted in mixture of acetonitrile-phosphate buffer (3:1, v/v; pH 8.5) and directly injected onto the chromatographic system. Continuous on-line derivatization and analysis of the compounds were successfully performed using in-tube elution of FMOC-Cl. The total time needed for derivatization and chromatographic analysis of the drug was 13 min. The assay was reliable and reproducible, with limit of quantification of 10 ng/ml. The described technique may offer significant advantages over existing off-line derivatization methods using FMOC-Cl.     

Optimal extraction conditions for high-performance liquid chromatographic determination of nucleotides in yeast

Different extraction methods of nucleotides from the yeast Saccharomyces cerevisiae were compared. A new extraction solution--formic acid saturated with 1-butanol--was found to be more effective than the commonly used solutions of trichloroacetic acid, perchloric acid, or formic acid alone. Using this solution the optimal extraction conditions were established. Nucleotide recovery was evaluated by adding standard nucleotides to the extraction medium and carrying them together with the cells through the whole extraction procedure. Nucleotides were separated and quantitated by high-performance liquid chromatography on an anion-exchange column.     

A new packing for separation of DNA restriction fragments by high performance liquid chromatography

TSK-GEL SW was found to be useful as a packing in high performance liquid chromatography for the separation of double-stranded DNA restriction fragments. DNA fragments smaller than 300 base pairs were separated as discrete peaks depending solely upon difference in chain length. The recovery of DNA fragments was higher than 90%.     

A thin-layer chromatographic method for separation of thymidine and deoxyuridine nucleotides

A thin-layer chromatographic method for the separation of thymidine and deoxyuridine nucleotides and nucleosides is described. This procedure involves the following sequence of steps: (i) Ion-exchange thin-layer chromatography to afford separation into fractions of increasing degree of phosphorylation, (ii) conversion of each fraction into an equivalent mixture of thymine and uracil through the combined actions of alkaline phosphatase and thymidine phosphorylase, and (iii) partition thin-layer chromatographic separation of thymine and uracil. A key feature of the method is the specificity afforded by the second step which converts only thymidine and deoxyuridine nucleotides and nucleosides to the corresponding pyrimidine bases. An application of the method to the study of [³H]deoxyuridine metabolism in L1210 cells, as well as the effect of methotrexate on this metabolism is also described.     

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.     

Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA.

We have developed a method to accurately determine (< 3% RSD) the complete major and modified base composition of a few micrograms of unlabeled DNA. The DNA samples were quantitatively hydrolyzed with DNase 1, Nuclease P1, and bacterial alkaline phosphatase. The resulting deoxyribonucleosides were directly separated in 70 min by reversed-phase high performance liquid chromatography with detection by ultraviolet absorption at 254 nm and 280 nm (RP-HPLC). The highly sensitive and selective dual wavelength quantitation greatly enhances the precision and accuracy of the chromatographic analysis. Contamination of DNA preparations with RNA does not interfere with the DNA analysis due to the high resolution of the chromatography. We have used this method for the quantitation of m5dCyd in 5 microgram of calf thymus and salmon sperm DNA in which the m5dCyd comprises only 1 to 2% of the total bases. This method should be a useful research tool in studies on various DNAs and DNA subfractions and should help to elucidate the functions of methylation of DNA.     

A new high performance liquid chromatographic method for quantification of atomoxetine in human plasma and its application for pharmacokinetic study

Atomoxetine is the first, non-stimulant alternative to other stimulant medications used for the treatment of Attention-Deficit/Hyperactivity Disorder (ADHD). Reported methods for the determination of atomoxetine include expensive liquid chromatography tandem mass spectrometry (LCMS) and high performance liquid chromatography (HPLC) with liquid scintillation counting (LSC) detection. Till date, no method has been reported in literature to determine atomoxetine using HPLC with UV detection. In this paper, we describe a new HPLC method for the determination of atomoxetine using liquid-liquid extraction with tertiary butyl methyl ether and UV detector. This method was found to be linear over the concentration range of 0.05-3.0 microg/ml. The limit of quantification was 0.05 microg/ml. Intra- and inter-day precision was <15% and accuracy was in the range of 95.67-108.80%. Stability studies showed that atomoxetine was stable in human plasma for short- and long-term period for sample preparation and analysis. This method was used for sample analysis in a pharmacokinetic study of atomoxetine (25mg) in five healthy adult female volunteers. The observed mean+/-S.D. pharmacokinetic parameters Cmax, Tmax and AUC(0-t) were 0.40+/-0.06 microg/ml, 3.40+/-0.42 h and 1.34+/-0.52 microg h/ml, respectively.     

A stability-indicating high performance liquid chromatographic (HPLC) assay for the simultaneous determination of atorvastatin and amlodipine in commercial tablets

A simple, rapid, precise and accurate isocratic reversed-phase stability-indicating HPLC method was developed and validated for the simultaneous determination of atorvastatin (AT) and amlodipine (AM) in commercial tablets. The method has shown adequate separation for AM, AT from their associated main impurities and their degradation products. Separation was achieved on a Perfectsil Target ODS-3, 5 microm, 250 mm x 4.6 mm i.d. column using a mobile phase consisting of acetonitrile-0.025 M NaH(2)PO(4) buffer (pH 4.5) (55:45, v/v) at a flow rate of 1 ml/min and UV detection at 237 nm. The drugs were subjected to oxidation, hydrolysis, photolysis and heat to apply stress conditions. The linearity of the proposed method was investigated in the range of 2-30 microg/ml (r=0.9994) for AT and 1-20 microg/ml (r=0.9993) for AM. The limits of detection were 0.65 microg/ml and 0.35 microg/ml for AT and AM, respectively. The limits of quantitation were 2 microg/ml and 1 microg/ml for AT and AM, respectively. Degradation products produced as a result of stress studies did not interfere with the detection of AT and AM and the assay can thus be considered stability-indicating.     

A sensitive high performance liquid chromatographic method for determining small amounts of glycosylproteins

We developed a simple isocratic high performance liquid chromatography (HPLC) method for the quantitative determination of 5-hydroxymethyl-2-furfuraldehyde (5-HMF) liberated by mild hydrolysis of small amounts of glycosyl proteins. The absorbance of hydrolysate components after HPLC separation was recorded at 280 nm. To detect substances possibly interfering with the 5-HMF peak we always recorded the ratio of the peak heights A280 nm/A254 nm which was a constant value of 4.4. For each sample the blank was obtained by reduction with NaBH4 before hydrolysis with oxalic acid 1 mol/l. The best NaBH4/protein ratio was found to be 4 mg/mg. With this method we measured the nonenzymatic glycosylation (glycation) as 5-HMF in samples with a protein concentration as low as 0.8 mg/ml. 5-HMF produced per milligram of protein was independent from protein concentration for a wide range (0.8-10 mg/ml). The mean coefficient of variation for within assay and between precision was 6.8 and 11.6%, respectively. The 5-HMF measured on plasma proteins from normal subjects (n = 7) was 0.16 +/- 0.04 nmol/mg. Protein from insulin-dependent diabetic patients was 0.31 +/- 0.07 nmol/mg. With this method we succeeded in detecting an excessive glycation of platelet membrane proteins in 13 type-I diabetic patients.     

A procedure for rapid extraction and high-pressure liquid chromatographic separation of the nucleotides and other small molecules from bacterial cells

A method for the extraction and separation of ribo- and deoxyribonucleosides and nucleotides from Salmonella typhimurium and Escherichia coli is described. Cells are harvested by a new rapid filtration method, and the small molecules are extracted with formic acid. The acid-extracted nucleotides and other uv-absorbing compounds are separated by boronate-affinity chromatography and by reverse-phase and ion-pair high-pressure liquid chromatography. The complete analysis uses the extract from 10 ml of log phase cells and separates the small molecule pool into about 170 components.     

A high pressure liquid chromatographic method for the separation and quantitation of water-soluble radiolabeled benzene metabolites

The glucuronide and sulfate conjugates of benzene metabolites as well as muconic acid and pre-phenyl- and phenylmercapturic acids were separated by ion-pairing HPLC. The HPLC method developed was suitable for automated analysis of a large number of tissue or excreta samples. p-Nitrophenyl [14C]glucuronide was used as an internal standard for quantitation of these water-soluble metabolites. Quantitation was verified by spiking liver tissue with various amounts of phenylsulfate or glucuronides of phenol, catechol, or hydroquinone and analyzing by HPLC. Values determined by HPLC analysis were within 10% of the actual amount with which the liver was spiked. The amount of metabolite present in urine following exposure to [3H]benzene was determined using p-nitrophenyl [14C]glucuronide as an internal standard. Phenylsulfate was the major water-soluble metabolite in the urine of F344 rats exposed to 50 ppm [3H]benzene for 6 h. Muconic acid and an unknown metabolite which decomposed in acidic media to phenylmercapturic acid were also present. Liver, however, contained a different metabolic profile. Phenylsulfate, muconic acid, and pre-phenylmercapturic acids as well as an unknown with a HPLC retention time of 7 min were the major metabolites in the liver. This indicates that urinary metabolite profiles may not be a true reflection of what is seen in individual tissues.     

A rapid high performance liquid chromatographic method for the separation of the alkaloid precursor L-tyrosine and six tetrahydroisoquinoline alkaloids of Papaver somniferum

A high performance liquid chromatographic (HPLC) method was developed for the qualitative and quantitative determination of L-tyrosine and six common tetrahydroisoquinoline alkaloids (papaverine, noscapine, sanguinarine, morphine, codeine and thebaine) of Papaver somniferum. The reversed phase HPLC method yields baseline separation of the alkaloids in 20 min and is achieved using a simple H₂O: MeOH linear gradient. Silanol effects commonly associated with the separation of such strongly basic compounds were minimized by the addition of the amine modifier, triethylamine, to the mobile phase.     

Analysis of the effects of ethanol, fructose and nicotinamide on the free nucleotides of rat liver using high performance liquid chromatography

1. Rat liver nucleotides were separated on a weak anion-exchange HPLC column. The characteristic nucleotide profile could be modified by treatment of the rats with ethanol, at both acute and chronic dosages, by fructose and by nicotinamide. 2. The major effect observed after ethanol administration was a decrease in the concentration of ATP with increases in the concentrations of AMP and other nucleotide monophosphates. 3. These changes gradually reverted to normal values over a 30 min period. 4. Ingestion of 1 or 5% ethanol for 4 weeks caused similar changes in the nucleotide profile of liver. 5. Fructose administration caused a dramatic but reversible decrease in the size of the entire nucleotide pool. 6. Rats given daily injections of nicotinamide exhibited greatly elevated concentrations of liver NAD+, whereas the other pyridine nucleotides were relatively unaffected. 7. The results are discussed in relation to the known effect on metabolism of the compounds tested.     

A method for separation and determination of cytokinin nucleotides from plant tissues

Recent progress in the study of cytokinin metabolism in plants indicates that quantitative analysis of cytokinin nucleotides is essential for elucidation of early steps of the biosynthetic pathway. However, traditional procedures for purification and quantification of cytokinin cannot discriminate the various nucleotides. We describe here a method for separation and determination of cytokinin nucleotides through a series of anion-exchange column chromatography steps followed by liquid chromatography-mass spectrometry. This method enabled us to analyze the amount of each species of cytokinin nucleotide in plant tissues.     

New high performance liquid chromatographic analysis of brain catecholamines

A new high performance liquid chromatography analysis has been developed for catecholamines in brain tissue. The method retains alumina separation of the catecholamines. Quantitative read-out is by direct liquid chromatography, replacing the tedious trihydroxyindole chemistry and fluorescence measurements. The analysis is rapid and accurate and agrees well with existing literature data. The equipment is inexpensive and the technique can be utilized for routine analyses after 1–2 weeks of practice. The method is directly applicable to whole small animal brains and, depending on the NE and DA levels, to dissected sub-portions.     

High performance liquid chromatographic determination of N-butyryl glucosamine in rat plasma

PURPOSE: A high performance liquid chromatography (HPLC) method for determination in plasma of N-butyryl glucosamine (GLBU), a highly water-soluble compound with no chromophore was developed. METHOD: To 100 muL of plasma containing GLBU was added fucose as internal standard. GLBU and fucose were derivatized using 1-phenyl-3-methyl-5-pyrazolone in the presence of sodium hydroxide at 70 degrees C for 30 min. The solution was neutralized with hydrochloric acid and the excess derivatizing reagent was extracted with chloroform. The aqueous layer was injected into an isocratic HPLC system consisting of an autoinjector, a single pump and a UV detector set at 245 nm. Two different 25 cm reversed phase columns were used, a 4 and a 10 microm C(18) columns. The mobile phase was a mixture of phosphate buffer (pH 7) and acetonitrile (80:20), which was run through a pump at a flow rate of 1.0 mL/min at ambient temperature. RESULTS: Derivatized fucose and GLBU appeared 24 and 28 min, and at 34 and 37 min using 4 and 10 microm columns, respectively. The assay was linear over the range of 0.2-200 microg/mL with a limit of quantification of 0.2 and 1 microg/mL for the 4 and 10 microm columns, respectively. The method was applied to the determination of GLBU in rat plasma after oral administration of 233 mg/kg of GLBU. CONCLUSION: The present assay is precise, and accurate with sufficient sensitivity for pharmacokinetic studies following therapeutically relevant doses.     

Development of high performance liquid chromatographic methods for the determination of cyadox and its metabolites in plasma and tissues of chicken

Cyadox (CYX) is an antimicrobial growth-promoter of the quinoxalines. It is highly effective on improving growth and feed conversion of chicken with little toxicity. For food safety concerns, HPLC-UV methods were developed for the sequential determination of CYX and its major metabolites, 1,4-bisdesoxycyadox (BDCYX) and quinoxaline-2-carboxylic acid (QCA), in plasma, muscle, liver, kidney and fat of chicken. For CYX and BDCYX, samples were subjected to a deproteinisation, a degrease and a liquid-liquid extraction. For QCA, samples were subjected to an alkali hydrolysis, a liquid-liquid extraction and a cation exchange column (AG MP-50 resin) clean-up. Analysis was performed on a RP-C18 column by UV detection with a gradient program of wavelength. Gradient elution was performed at a flow of 1mL/min. The limits of quantification for CYX, BDCYX and QCA in plasma and tissues were 0.025microg/g, and 0.002microg/g for QCA in muscle. The recoveries of three compounds in plasma and tissues were 70-87% with inter-day relative standard deviation (R.S.D.) less than 10%. An animal experiment was performed to show the applicability of the present methods in real biological samples, which demonstrated a satisfactory applicability since all compounds could be detected nearly in all tissues. The present methods were highly sensitive and accurate, and could therefore be useful in pharmacokinetic and residue studies for cyadox in chicken. The developed methods will be further applied in the residue screening of cyadox and its metabolites in chicken.     

High-performance liquid chromatographic separation of aprotinin-like inhibitors and their determination in very small amounts

A reversed-phase high-performance liquid chromatographic method for the determination and quantitative recovery of fully active aprotinin (the basic pancreatic trypsin inhibitor or Kunitz inhibitor) and aprotinin-like inhibitors in amounts down to 0.5 μg is reported. The method, which allows separation of aprotinin isoinhibitors characterized by small differences in the primary structure with respect to aprotinin itself, appears to be suitable for the quantitation and identification of aprotinin-like inhibitors in human biological fluids, in which they appear to be present at very low levels.     

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.