Dual-column high-performance liquid chromatographic urinary organic acid profiling

Two-dimensional high-performance liquid chromatography (HPLC) is increasingly used for the separation and identification of compounds in biological matrices. Conventional two-dimensional HPLC involves either heart-cutting or column-switching techniques. These techniques work well but are very time consuming when the analysis involves many compounds and requires more than a few minutes to completely elute from the column. We have modified the column-switching technique by utilizing two columns in series, an Aminex HPX-87H organic acid column followed by a 15-cm 5-μm C₁₈ analytical column. Both columns are compatible with the isocratic pH 2.5 H₂SO₄ mobile phase employed in the organic acid profiling. The dual-column system affords better resolution of urinary organic acids than does either column separately. Reversing the column order does not dramatically affect the elution pattern (peak shape, peak height, and R_f values are approximately the same). The dualcolumn HPLC configuration works well as a rapid means of screening urinary carboxylic acids prior to subsequent definitive analyses of abnormal samples.     

Stereospecific high-performance liquid chromatographic assay of metoprolol

A valid, sensitive high-performance liquid chromatographic technique is reported for the separation of the two enantiomers of metoprolol in human plasma. The procedure involves pre-column derivatization with the homochiral reagent S-(+)-1-(1-naphthyl)-ethyl isocyanate. Once formed, the diastereomers are separated using normal-phase high-performance liquid chromatography. Fluorescence detection (220 nm excitation; no emission filter) was utilized, resulting in baseline resolution (R_s > 1.5). The peaks corresponding to metoprolol enantiomers were free from interference throughout the examined range of 5–500 ng/ml; accuracy and precision were within approximately 10%. Analysis of a plasma sample collected from a healthy volunteer demonstrated that the assay is applicable to clinical studies.     

Extraction, pre-high-performance liquid chromatographic purification, and high-performance liquid chromatographic analysis of plant nucleotides

Problems encountered in obtaining reliable analytical data by HPLC for the free nucleotide constituents of plant tissues are considered and methods of overcoming them experimentally assessed. Major problems include suppression of residual phosphatase activity during extraction, and removal of pigments, phenolics, alkaloids, and other uv-absorbing nonnucleotides, prior to HPLC. An optimal combination of extraction and pre-HPLC purification techniques is discussed which, in combination with HPLC by anion exchange, yields quantitatively reliable data. The optimized procedure involves extraction with a monophasic mixture of methanol: chloroform:formic acid:water and purification of the nucleotide extract by a batch treatment with poly-N-vinylpyrrolidone, followed by ligand-exchange chromatography. The main HPLC separation uses mu Bondapak NH2 in a linear phosphate gradient and gives good resolution of all the commonly occurring plant nucleotides in a single chromatographic run.     

Rapid high-performance liquid chromatographic assay for atovaquone

A rapid high-performance liquid chromatography assay has been developed for the drug atovaquone, which is currently being used to treat Pneumocystis carinii pneumonia and Taxoplasma gondii encephalitis associated with the acquired immunodeficiency syndrome (AIDS). Protein is precipitated from plasma with acetonitrile-aqueous 1% acetic acid (85:15). The supernatant is assayed on a C₆ column using methanol-10 mM triethylamine in aqueous 0.2% trifluoroacetic acid (76:24) with detection at 254 nm. The working assay range was 0.5 to 50 μg/ml. Recovery was 97% and the between-day coefficients of variation were 2.1% at 50 μg/ml and 10.3% at 1 μg/ml. A number of drugs commonly used to treat AIDS and its complications did not interfere with the assay.     

Determination of 3,4-dihydroxyphenyl glycol in plasma by gas chromatography-mass spectrometry and high-performance liquid chromatography methods

Several modifications of GC-MS and HPLC methods for plasma level DHPG have been described. The effects of storage temperature and stabilizing agents on DHPG stability have been studied. The stabilizing agent has been found to play a more important role than low-temperature storage in preventing DHPG from decomposition during sample storage. A specific and sensitive GC-MS method (electron impact) has been established using stable isotope-labeled DHPG as an internal standard. HPLC has been improved by modifying the conditions, resulting in a good separation of DHPG and internal standard from solvent front and other early eluting compounds. Comparison of the GC-MS and HPLC procedures demonstrates a strong correlation between these two methods.     

Study of the metabolism of pyrazinamide using a high-performance liquid chromatographic analysis of urine samples

A reversed-phase high-performance liquid chromatographic method was developed for the simultaneous determination of pyrazinamide and its metabolites in urine. Study of the metabolism of pyrazinamide by this method demonstrated that 5-hydroxypyrazinamide excretion was compatible with pyrazinoic acid excretion and allopurinol decreased in vivo conversion of pyrazinamide to 5-hydroxypyrazinamide and blocked that of pyrazinoic acid to 5-hydroxypyrazinoic acid.     

Derivatization and high-performance liquid chromatographic analysis of pentaazapentacosane pentahydrochloride

A rapid high-performance liquid chromatographic method for determination of the dansyl derivative of pentaazapentacosane (PAPC) pentahydrochloride has been developed. The chromatographic system uses a reversed-phase C₈ column, a mobile phase of acetic acid buffer and acetonitrile and UV detection. The dansylation conditions were optimized with a pH of 11.0 and a 20-fold dansyl chloride excess. The yield of dansyl PAPC increased 10-fold as the reaction pH was changed from 9.5 to 10.5. Under derivatization conditions of pH 8.5–11.0 and 1–30-fold excess dansyl chloride only perdansyl PAPC was found.     

Direct high-performance liquid chromatographic enantioseparation of beta-lactam stereoisomers

High-performance liquid chromatographic methods were developed for the separation of the enantiomers of 12 beta-lactams. Direct separations were performed on chiral stationary phases (CSPs) containing cellulose-tris-3,5-dimethylphenyl carbamate (Chiralcel OD-RH and OD-H columns), the macrocyclic glycopeptide antibiotic teicoplanin (Chirobiotic T column), or teicoplanin aglycone (Chirobiotic TAG column) as the chiral selector. It was clearly established that, with teicoplanin-based columns, the teicoplanin aglycone was most often responsible for the enantioseparation of the beta-lactams. The difference in enantioselective free energy between the aglycone CSP and the teicoplanin CSP was in the range between 0.02 and 0.97 kJ mol(-1) for these beta-lactam stereoisomer separations. The separations were carried out with high selectivity and resolution, and the method was therefore suitable for monitoring of the enantiomeric excess after chiral synthesis. The Chirobiotic and Chiralcel columns appear to be highly complementary to one another. The best separation of this class of beta-lactam compound could be obtained using the Chirobiotic TAG in the polar-organic mode plus the Chiralcel OD-H in the normal-phase mode. The elution sequence was also determined.     

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.     

Automated high-performance liquid chromatographic assay of enzymatic activities

High-performance liquid chromatography (HPLC) is a powerful technique which enables a reliable and quantitative determination of enzyme activities. The purpose of the work reported here was to develop an automatic assay of enzymatic activity. Using an automatic sample processor and injector, a program was developed which allows the complete automation of each step of analysis (calibration, enzymatic reaction, HPLC determination). This program can be adapted to different experimental requirements as each step can be performed independently and each input (time, volume, number of standards) is made by answering questions asked by instrument. Using this approach both kinetic and single-point determinations can be carried out, and in the latter case different samples can be analysed sequentially. This paper reports the automated analysis of trypsin.     

Anion-exchange high-performance liquid chromatographic analysis of inositol phosphates

The analysis of inositol phosphates by anion-exchange HPLC is described. The method employs a citrate buffer gradient to resolve several inositol phosphates including inositol 1-phosphate, inositol 1,4-bisphosphate (IP2), and inositol 1,4,5-trisphosphate (IP3), as well as some of the isomers of these compounds. Since the buffer system does not contain any phosphate, we can use a phosphate assay to examine the chromatographic behavior of phosphate-containing compounds. The method shows good resolution and recovery (greater than 95% for IP2 and IP3). Total analysis time, including reequilibration, is about 90 min. In addition, an isocratic system that can rapidly (less than 10 min) measure IP3 is described. The HPLC system was used to characterize inositol phosphate turnover in thrombin-stimulated platelets and formylmethionyl-leucyl-phenylalanine-stimulated HL-60 cells.     

Validation of high-performance liquid chromatographic assay methods for the analysis of carboplatin in plasma ultrafiltrate

Validation of two HPLC assays for the quantitation of carboplatin in human plasma ultrafiltrate is described. Both assay methods employed a YMC ODS-AQ 3.9×150 mm (3 μm) column for the chromatographic separation. The first method utilized direct UV detection, the second method utilized UV detection following post-column derivatization with sodium bisulfite. Structural analogues of carboplatin were synthesized and used as internal standards for the assays. With direct UV detection, sample clean-up using solid-phase extraction on amino cartridges was required prior to injection, with extraction recoveries ranging from 80 to 90%. This extraction procedure was not necessary with the post-column reaction method, which employed a more selective analytical wavelength. Unfortunately, instability of the post-column reagent was a problem and led to greater variability in predicted concentration values. For standard curves, a weighted (1/y²) regression approach was used for plots of peak area or peak height ratio (carboplatin/internal standard) vs. carboplatin concentration. The limit of detection of both assays was 0.025 μg/ml and both were validated for carboplatin concentrations from 0.05 to 40 μg/ml. Accuracy and precision data were generated using three batches of validation samples, each batch consisting of a standard curve and five sets of quality control samples. Stability of carboplatin in blood, plasma, plasma ultrafiltrate, and reconstituted extracts was evaluated. The assay methods were employed for the pharmacokinetic analysis of blood samples drawn from a pediatric patient that received a 400 mg/m² dose of carboplatin.     

Assay of moguisteine metabolites in human plasma and urine: conventional and chiral high-performance liquid chromatographic methods

Moguisteine is a novel peripheral non-narcotic antitussive agent. Pharmacokinetic studies in animal and in man showed that no unchanged drug is present in plasma, urine and faeces after oral administration. The main active metabolite, M1, is the free carboxylic acid of moguisteine, which maintains a stereogenic centre and consists of R(+)-M1 and S(−)-M1 enantiomers. M1 is partly metabolized to M2, its sulfoxidation derivative. A conventional HPLC method is described for the simultaneous determination of M1 and M2 in human plasma and urine after administration of therapeutic moguisteine doses. Plasma samples, previously acidified with phosphoric acid, are extracted with dichloromethane; urine samples are analyzed after appropriate dilution with methanol. Chromatography is performed using a Lichrosorb RP2 column and a linear gradient. M1 enantiomers can be determined in plasma extracts and urine samples by a chiral HPLC method using a β-cyclodextrin column. The analytical characteristics of both HPLC procedures proved to be adequate to analyze samples of subjects treated with therapeutic doses of moguisteine during clinical pharmacokinetic studies.     

Paired-ion high-performance liquid chromatographic assay for plasma choline

Choline was isolated from deproteinized plasma by cation-exchange chromatography. Isolated choline was directly converted to the 3,5-dinitrobenzoate derivative and was analyzed by paired-ion high-performance liquid chromatography with UV detection at 254 nm. An internal standard, 3-hydroxy-N,N,N-trimethylpropanaminium iodide was used for quantitation of plasma choline.Linearity was achieved from 1–500 nmole/ml with a reproducibility of ± 6%. Plasma choline concentrations below 1 nmole/ml could not be accurately measured while plasma choline concentrations in the μmole/ml range deviated from linearity.     

Characterization of human alcohol dehydrogenase isoenzymes by high-performance liquid chromatographic peptide mapping

Human liver alcohol dehydrogenase (ADH, EC 1.1.1.1) is a large and heterogeneous family of isoenzymes and the high-performance liquid chromatographic peptide mapping technique which was developed here recognizes differences and similarities between them. Isoenzymes were S-carboxymethylated, digested with trypsin, and the mixtures of tryptic peptides fractionated by reverse-phase gradient chromatography on octadecylsilane columns, using perchlorate-phosphate buffer and acetonitrile as eluants. The resultant peptide maps were reproducible, showing great similarities between the αβγ-ADH isoenzymes (now called Class I) on the one hand and remarkable differences between these and both the π- and χ-ADH isoenzymes (now called Class II and III, respectively) on the other. This implies that these three isoenzyme groups have characteristic primary structures which correspond to their typical substrate specificities and kinetics.     

Analysis of tissue glycoprotein sugar chains by two-dimensional high-performance liquid chromatographic mapping

Excellent separation of 45 pyridylamino derivatives of oligosaccharides were achieved by the two-dimensional combination of reversed-phase and size-fractionation high-performance liquid chromatography. The sugar chains of brain glycoproteins were derivatized into a mixture of pyridylamino-oligosaccharides from lyophilized brain tissue without any purification steps, and they were well separated by the system used. The pattern obtained was reproducible, and inter-individual variation was negligible. This finding demonstrated the possibility that this method could be applied to the detection of differences in the structure of glycoprotein sugar chains in crude preparations.     

Tissue extraction and high-performance liquid chromatographic determination of ketoprofen enantiomers

Local transcutaneous delivery of non-steroidal anti-inflammatory drugs avoids gastrointestinal side effects and concentrates drugs in the intended tissues. An extraction and HPLC method was developed for ketoprofen in skin, fascia and muscle. Tissue samples were homogenized in NaHCO₃. After methylene chloride removal of lipids, the aqueous layer was acidified with HCl and back extracted into isooctane/isopropanol. Ketoprofen was derivatized with ethylchloroformate/S-(−)-α-phenylethylamine in triethylamine, then detected by HPLC. Ketoprofen recovery was linear (1–33 μg/g) and was detected in these tissues following in vivo cathodic iontophoresis (160 mA*min). This represents the first non-radioactive method for determination of ketoprofen in tissues following transcutaneous iontophoresis.