Determination of urinary glucuronide conjugates by high-performance liquid chromatography with pre-column fluorescence derivatization

A simple and highly sensitive high-performance liquid chromatographic method for the direct determination of urinary glucuronide conjugates is described. The method is based on the direct derivatization of the glucuronic acid moiety in glucuronide conjugates with 6,7-dimethoxy-1-methyl-2 (1 H)-quinoxalinone-3-propionylcarboxylic acid hydrazide. The derivatization reaction proceeds in aqueous solution in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at 0–37°C. The resulting fluorescent derivatives are separated on a C₁₈ column using methanol—acetonitrile—0.5% triethylamine in water (1:1:2, v/v) as mobile phase, and are detected spectrofluorimetrically at 445 nm with excitation at 367 nm. The detection limits (signal-to-noise RATIO = 3) for the glucuronides are 13–48 fmol for an injection volume of 10 μl (130–480 fmol per 5 μl of human urine). The method was applied to the measurement of etiocholanorone-3-glucuronide and androsterone-3-glucuronide in human urine. The method is simple and rapid without conventional liquid—liquid extraction of the glucuronides from urine.     

Determination of ethambutol in plasma by high-performance liquid chromatography after pre-column derivatization

A new HPLC assay using UV detection (200 nm) was developed to determine ethambutol (EMB) concentrations in plasma. Following extraction (0.1 ml plasma) with chloroform, EMB and octylamine (used as internal standard) were derivatized with phenylethylisocyanate. Quantitation in plasma was achieved at 200 nm. There were no interferences from endogenous compounds. Intra- and inter-day variabilities were lower than 5.2 and 7.6%, respectively. The limit of quantitation of the method was 0.2 μg/ml. In plasma, ethambutol was found to be stable for at least one month when samples were stored at −20°C. This assay was applied to the therapeutic monitoring of EMB concentrations in 19 patients suffering from tuberculosis.     

Measurement of allantoin in urine and plasma by high-performance liquid chromatography with pre-column derivatization

A method is reported for determination of allantoin in urine and plasma based on high-performance liquid chromatography (HPLC) and pre-column derivatization. In the derivatization procedure, allantoin is converted to glyoxylic acid which forms a hydrazone with 2,4-dinitrophenylhydrazine. The hydrazone appears as syn and anti isomers at a constant ratio. These derivatives are separated by HPLC using a reversed-phase C₁₈ column from hydrazones of other keto acids possibly present in urine and plasma and then monitored at 360 nm. All components were completely resolved in 15 min. Both the reagents and derivatization products are stable. Recovery of allantoin added to urine and plasma was 95 ± 3.7% (n = 45) and 100 ± 7.5% (n = 64), respectively. The lowest allantoin concentration that gave a reproducible integration was 5 μmol/l. The between-assay and within-day coefficients of variation were 2.8 and 0.6%, respectively.     

Determination of busulfan in human plasma using high-performance liquid chromatography with pre-column derivatization and fluorescence detection

A rapid, sensitive and reproducible high-performance liquid chromatographic assay for busulfan in human plasma was developed. After extraction of plasma samples with acetonitrile and methylene chloride, busulfan and the internal standard [1,5-bis(methanesulfonyloxy)pentane] were derivatized with 8-mercaptoquinoline to yield fluorescent compounds which were detected with a fluorescence detector equipped with filters of 360 nm (excitation) and 425 nm (emission). Calibration graphs showed a linear correlation (r>0.9990) over the concentration range of 20–2000 ng/ml. The recovery of busulfan from plasma standards was 70±5%. The detection and quantification limits for busulfan in plasma samples were established at 9 ng/ml and 20 ng/ml, respectively. The intra- and inter-assay variations were lower than 8% and 10%, respectively. The applicability of the method was verified by analyzing the plasma concentrations of busulfan in a patient to whom it was administered orally on two different days.     

Determination of penicillin G in bovine plasma by high-performance liquid chromatography after pre-column derivatization

A simple, selective, and sensitive liquid chromatographic method with ultraviolet detection was developed for the analysis of penicillin G in bovine plasma. The assay utilizes a simple extraction of penicillin G from plasma (with a known amount of penicillin V added as internal standard) with water, dilute sulphuric acid and sodium tungstate solutions, followed by concentration on a conditioned C₁₈ solid-phase extraction column. After elution with 500 μl of elution solution, the penicillins are derivatized with 500 μl of 1,2,4-triazole—mercuric chloride solution at 65°C for 30 min. The penicillin—mercury mercaptide complexes are separated by reversed-phase liquid chromatography on a C₁₈ column. The method, which has a detection limit of 5 ng/ml (ppb) in bovine plasma, was used to quantitatively measure the concentrations of penicillin G in plasma of steers at a series of intervals after the intramuscular administration of a commercial formulation of procaine penicillin G.     

Fluorometric determination of total vitamin C in whole blood by high-performance liquid chromatography with pre-column derivatization

A reliable and semi-automated high-performance liquid chromatographic (HPLC) method is described for the determination of total vitamin C in whole blood. After deproteinization of whole blood and enzymatic oxidation of l-ascorbic acid to dehydro-l-ascorbic acid, the latter is condensed with o-phenylenediamine to its quinoxaline derivative. This derivative is separated on a reversed-phase HPLC column and detected fluorometrically. Total vitamin C in whole blood can be determined in concentrations as low as 0.2 μmol/l.Special attention was paid to the stability of vitamin C in whole blood and of its quinoxaline derivative in the extract. Results of our investigations showed that total vitamin C in whole blood is stable for eight days at −20°C, provided ethyleneglycol-bis-(β-amino-ethyl ether)-N,N,N′,N′-tetraacetic acid and glutathione are immediately added to the blood sample. The quinoxaline derivative of vitamin C in the blood extract is stable for at least 24 h if stored in the dark at 4°C.Routine vitamin C determinations can be carried out in a series of 100 samples within 48 h. The within-assay and between-assay coefficients of variation were 3.7% and 4.6%, respectively. The between-assay analytical recovery of l-ascorbic acid added to whole blood samples was 97.0 ± 7.0% (mean ± S.D.). Reference values of vitamin C in whole blood of normal healthy Dutch adults were found in the range 20–80 μmol/l.     

Simultaneous determination of N-acetylaspartylglutamate and N-acetylaspartate in rat brain homogenate using high-performance liquid chromatography with pre-column fluorescence derivatization

Simultaneous determination method of N-acetyl-l-aspartyl-l-glutamate (NAAG), an endogenous agonist at type 3 metabotropic glutamate receptor, and its degradation product, N-acetyl-l-aspartate (NAA) was developed by using reversed-phase high-performance liquid chromatography (HPLC) with pre-column fluorescence derivatization using 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole. The detection limits of NAAG and NAA were approximately 12 and 34 fmol on the column, respectively (signal to noise ratio 3). The proposed HPLC method was applied to determine NAAG and NAA simultaneously in the rat brain homogenate. Both concentrations of NAAG and NAA in the male rat cerebrum (13 weeks old) were 5.7+/-0.30 and 2.1 x 10(2)+/-9.2 nmol/mg protein, respectively (n=6), while those in the hippocampus were 6.8+/-0.48 and 1.9 x 10(2)+/-8.5 nmol/mg protein, respectively (n=5). Hippocampal NAA concentration was significantly increased in the ketamine-treated rats as compared to the control rats (p<0.01).     

Validation of the determination of amino acids in plasma by high-performance liquid chromatography using automated pre-column derivatization with o-phthaldialdehyde

A sensitive and reproducible fully automated method for the determination of amino acids in plasma based on reversed-phase high-performance liquid chromatography and o-phthaldialdehyde pre-column derivatization is described. A 5-μm Spherisorb ODS 2 column (125 × 3 mm I.D.) was selected for routine determination. Over 40 physiological amino acids could be determined within 49 min (injection to injection) and 48 samples could be processed unattended. The coefficients of variation for most amino acids in plasma were below 4%. We were also able to measure trace amounts of amino acids in plasma normally not detected in a routine analysis. The results obtained with the method described compared favourably with those of conventional amino acid analysis (r = 0.997) and were in excellent agreement with those of other laboratories (r = 0.999).     

Simultaneous determination of primary and secondary d- and l-amino acids by reversed-phase high-performance liquid chromatography using pre-column derivatization with two-step labelling method

This work describes a method for the simultaneous determination of primary d- and l-amino acids and secondary amino acids such as d- and l-proline. In order to remove interferences in the simultaneous determination of primary and secondary amines, the primary amines were derivatized with o-phthalaldehyde/N-acetyl-l-cysteine (OPA/NAC) and subsequently with 1-(9-fluorenyl)ethyl chloroformate (FLEC) for secondary amines, in a pre-column separation derivatization technique. These fluorescent diastereomers of the amino acids were obtained within 3 min at room temperature and determined simultaneously by changing wavelengths during analysis in a single eluting run in the high-performance liquid chromatography column. This method, referred to as the “two-step labelling method,” is effective for the simultaneous determination of d- and l-amino acids.     

Sensitive microanalysis of gabapentin by high-performance liquid chromatography in human serum using pre-column derivatization with 4-chloro-7-nitrobenzofurazan: Application to a bioequivalence study

Most of the published methods for analysis of gabapentin, an antiepileptic agent, in human serum require automated o-phthalaldehyde derivatization of the drug and immediate injection of the unstable derivatives formed. A new, very sensitive and simple high-performance liquid chromatographic method for quantitation of the drug in human serum using 4-chloro-7-nitrobenzofurazan (NBD-Cl) as a fluorescent labeling agent is presented. In this method the sensitivity was significantly improved and the limit of quantification of 0.002 microg/ml was obtained using 100 microl serum sample and 10 microl injection. However, the LOQ can be improved by increasing the sampling volume. The procedure involved protein precipitation of serum by acetonitrile followed by derivatization with NBD-Cl. Amlodipine was used as internal standard and chromatographic separation was performed on a Shimpack CLC-C18 (150 mm x 4.6 mm) column. The fluorescence derivative of the drug was monitored at excitation and emission wavelengths of 470 and 537 nm, respectively. A mobile phase consisting of methanol and sodium phosphate buffer (0.05 M; pH 2.5) containing 1 ml/l triethylamine (65:35, v/v) was used. The calibration curve was linear over the concentration range of 0.002-15 microg/ml. No interferences were found from commonly co-administrated antiepileptic drugs. The method was applied in a randomized cross-over bioequivalence study of two different gabapentin preparations in 24 healthy volunteers.     

Measurement of free amino acid levels in ultrafiltrates of blood plasma by high-performance liquid chromatography with automatic pre-column derivatization

Although there are many techniques available for the analysis of amino acids, deproteinization is still one of the major problems in the analysis of amino acids in physiological fluids. The method used to prepare the plasma and to remove the plasma protein has a marked effect on the final results. The most widely used method of deproteinization is precipitation with 5-sulphosalicylic acid followed by centrifugation to remove the precipitated protein. We have not had success in using this deproteinization agent for the analysis of plasma amino acids by a high-performance liquid chromatographic method with automatic pre-column o-phthaldialdehyde—3-mercaptopropionic acid and 9-fluorenylmethyl chloroformate derivatization because of the adverse effect of the sulphosalicyclic acid supernatant on the quantitation and separation. Ultrafiltration was used as an alternative method for the preparation of plasma samples in this experiment. The results were satisfactory for the analysis of plasma amino acids in 1500 samples during a period of four years. Some factors that might influence the results of the ultrafiltration were investigated.     

Analysis of plasma catecholamines by high-performance liquid chromatography with fluorescence detection: simple sample preparation for pre-column fluorescence derivatization

Analysis of plasma catecholamines (norepinephrine, epinephrine and dopamine) by high-performance liquid chromatography using 1,2-diphenylethylenediamine as a fluorescent reagent is described. We have developed an automatic catecholamine analyser, based on pre-column fluorescence derivatization and column switching. The analysis time for one assay was 15 min. The correlation coefficients of the linear regression equations were greater than 0.9996 in the range 10–10 000 pg/ml. The detection limit, at a signal-to-noise ratio of 3, was 2 pg/ml for dopamine. A new method of sample preparation for the pre-column fluorescence derivatization of plasma catecholamines was used. In order to protect the catecholamines from decomposition, an ion-pair complex between boric acid and the diol group in the catecholamine was formed at a weakly alkaline pH. The stabilities of plasma catecholamines were evaluated at several temperatures. After complex formation, the catecholamines were very stable at 17°C for 8 h, and the coefficients of variation for norepinephrine, epinephrine and dopamine were 1.2, 4.2 and 9.3%, respectively.     

Extraction and measurement of pamidronate from bone samples using automated pre-column derivatization, high-performance liquid chromatography and fluorescence detection

A method for the determination of 3-amino-1-hydroxylpropylidene-1,1-bisphosphonic acid (pamidronate) in bone samples is described. This method combines and modifies parts of previous procedures. Pamidronate is extracted from finely ground bone with dilute hydrochloric acid. Amine-containing contaminants are removed by co-precipitation of pamidronate with calcium. Excess calcium is removed with EDTA and an ion-exchange resin. Pamidronate is automatically derivatized at the primary amine and quantified by high-performance liquid chromatography with fluorescence detection. The method assay was linear in the concentration range 7.5–600 ng/mg bone (20–1000 pmol/mg). The imprecision for repeat analyses were 16.5 and 7.8%, at pamidronate levels of 7.5 and 600 ng/mg bone, respectively. The method has been used to analyze bone samples from pharmacokinetic animal studies involving both acute and chronic dosages.     

Determination of clarithromycin in human serum by high-performance liquid chromatography after pre-column derivatization with 9-fluorenylmethyl chloroformate: application to a bioequivalence study

A sensitive liquid chromatographic method for the analysis of clarithromycin, a macrolide antibiotic, in human serum using pre-column derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) is described. The method involved liquid-liquid extraction of the drug and an internal standard (amantadine) followed by pre-column derivatization of the analytes with FMOC-Cl. A mixture of 0.05 M phosphate buffer containing triethylamine (2 mL L(-1); pH 3.8) and methanol (17:83, v/v) was used as mobile phase and chromatographic separation was achieved on a Shimpack CLC-ODS column. The eluate was monitored by a fluorescence detector with respective excitation and emission wavelengths of 265 and 315 nm. The analytical method was linear over the concentration range of 0.025-10 microg mL(-1) of clarithromycin in human serum with a limit of quantification of 0.025 microg mL(-1). The assay is sensitive enough to measure drug levels obtained in human single dose studies. In the present method, sensitivity and run time of analysis have been improved, and successfully applied in a bioequivalence study of three different clarithromycin preparations in 12 healthy volunteers.     

Stereoselective high-performance liquid chromatography determination of propranolol and 4-hydroxypropranolol in human plasma after pre-column derivatization

A stereoselective reversed-phase HPLC assay to quantify S-(−) and R-(+) enantiomers of propranolol and 4-hydroxypropranolol in human plasma was developed. The method involved liquid–liquid extraction for sample clean-up and employed 2,3,4,6-tetra-O-acetyl-β-glucopyranosyl isothiocyanate as a pre-column chiral derivatization reagent. The internal standard used was 4-methylpropranolol. The derivatized products were separated on an Altex C₁₈ column using a mixture of acetonitrile–water–phosphoric acid–triethylamine (58:42:0.1:0.06 and 50:50:0.15:0.06, v/v, for propranolol and 4-hydroxypropranolol, respectively) as mobile phase. The detection of propranolol derivatives was made at λ_ex=280 nm and λ_em=325 nm, and the corresponding 325 and 400 nm were used for 4-hydroxypropranolol derivatives. The assay was linear from 1 to 100 ng/ml and from 2 to 50 ng/ml using 0.5 ml of human plasma for propranolol and 4-hydroxypropranolol enantiomers, respectively. The present assay is used to quantify the enantiomers of propranolol and 4-hydroxypropranolol, respectively, in human plasma for pharmacokinetic studies.     

Determination of l-carnitine,acetyl-l-carnitine and propionyl-l-carnitine in human plasma by high-performance liquid chromatography after pre-column derivatization with 1-aminoanthracene

A new sensitive high-performance liquid chromatographic procedure for the determination of l-carnitine (LC), acetyl-l-carnitine (ALC) and propionyl-l-carnitine (PLC) in human plasma has been developed. Precolumn derivatization with 1-aminoanthracene (1AA), performed in phosphate buffer in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) as catalyst, is involved. The fluorescent derivatives were isocratically separated on a reversed-phase column (C₁₈). The eluate was monitored with a fluorimetric detector set at 248 nm (excitation wavelength) and 418 nm (emission wavelength). Because of the presence of endogenous carnitines, the validation was performed using dialyzed plasma. The identity of the derivatized compounds was assessed by mass spectrometry and the purity of the chromatographic peaks was confirmed by HPLC-tandem mass spectrometry. The limits of quantitation were 5 nmol/ml for LC, 1 nmol/ml for ALC and 0.25 nmol/ml for PLC. The recovery of the extraction procedure was in the range 82.6%–95.4% for all 3 compounds. Good linearity (R≈0.99) was observed within the calibration ranges studied: 5–160 nmol/ml for LC, 1–32 nmol/ml for ALC and 0.25–8 nmol/ml for PLC. Precision was in the range 0.3–16.8% and accuracy was always lower than 10.6%.     

Determination of biogenic amines in beer with pre-column derivatization by high performance liquid chromatography

Eighteen samples of commercially available Chinese beer were analyzed in order to determine the content of biogenic amines. The method involves pre-column derivatization of the amines with 4-chloro-3,5-dinitrobenzotrifluoride (CNBF) and subsequent analysis by RP-HPLC (reversed phase-high performance liquid chromatography) with diode array detection. The labeled biogenic amines were separated on a Kromasil C18 column (250 mm × 4.6 mm, 5 μm) at room temperature and UV detection was applied at 254 nm. The separation of seven labeled biogenic amines was achieved within 22 min by elution acetonitrile and HAc–NaAc buffers. The method linearity, calculated for each biogenic amine, has a correlation coefficient higher than 0.9925, in concentrations ranging from 2.9 μmol L^?1 to 565 μmol L^?1. Detection limits of biogenic amines were 0.056–0.87 μmol L^?1, at a signal-to-noise ratio of 3. The proposed method has been applied to the quantitative determination of spermine, phenethylamine, spermidine, histamine, tyramine, tryptamine and putrescine in beer with recoveries of 91.9–103.1% and R.S.D. of 2.86–5.63%. Quantitation is relative to external standards. The results showed that each kind of beer examined contained at least three biogenic amines. Putrescine, histamine and tyramine were detected in all samples. Spermidine was detected in 89% of the beers. Spermine, tryptamine and phenylethylamine occurred in 78%, 61% and 44% of the beers examined, respectively. These levels were below the level that may elicit direct adverse reactions for most consumers.     

Quantitative determination of the macrolide antibiotics erythromycin, roxithromycin, azithromycin and clarithromycin in human serum by high-performance liquid chromatography using pre-column derivatization with 9-fluorenylmethyloxycarbonyl chloride and fluorescence detection

A validated, highly sensitive and precise high-performance liquid chromatographic (HPLC) method for the determination of the macrolides erythromycin, azithromycin, clarithromycin and roxithromycin in human serum is described. A diethyl ether extract, obtained from serum using a saturated sodium carbonate solution, was treated with 9-fluorenylmethyloxycarbonyl chloride (FMOC–Cl) for 40 min at 40°C and chromatographed on a base-deactivated octadecyl column, maintained at 50°C during elution, using an eluent composed of acetonitrile–hydrogenphosphate buffer, pH 7.5, with 0.125% triethylamine (3:2, v/v). Fluorescence detection was used at an excitation wavelength of 255 nm and an emission wavelength of 315 nm. Erythromycin, clarithromycin, roxithromycin and azithromycin were found to have retention times of 8.8, 15.7, 17.1 and 20.7 min, respectively. Recoveries ranging from 93 to 104% were found with reproducibility coefficients of variation of 1.1–5%. Mean correlation coefficients of 0.9997, 0.9998, 0.9996 and 0.9994 were found for the linear calibration curves (n=2) of erythromycin (0.320–16.1 mg/l), roxithromycin (3.24–19.4 mg/l), clarithromycin (0.190–19.4 mg/l) and azithromycin (0.0988–4.94 mg/l), respectively.     

Determination of polyoxyethyleneglycerol triricinoleate 35 (Cremophor EL) in plasma by pre-column derivatization and reversed-phase high-performance liquid chromatography

A sensitive and selective reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of polyoxyethyleneglycerol triricinoleate 35 (Cremophor EL; CrEL), which requires only microvolumes (20 μl) of plasma, has been developed and validated. The procedure is based on saponification of CrEL in alcoholic KOH, followed by extraction of the released fatty acid ricinoleic acid with chloroform and derivatization with 1-naphthylamine. Margaric acid was used as the internal standard. The products are separated using an HPLC system consisting of an analytical column packed with Spherisorb ODS-1 material and a mobile phase of methanol-acetonitrile-10 mM potassium phosphate buffer pH 7.0 (72:13:15, v/v). Detection was executed by UV absorption at 280 nm. The lower limit of quantitation and the lower limit of detection in plasma are 0.01 and 0.005% (v/v) of CrEL, respectively. The percentage deviation and precision of the procedure, over the validated concentration range of 0.01 to 1.0% (v/v) of CrEL in plasma, are ≤8.0% and ≤ 6.6%, respectively. Compared to the previously described bioassay, the presented HPLC method possesses superior sensitivity and reliability. Preliminary pharmacokinetic studies of CrEL in mice and patients receiving paclitaxel formulated in CrEL have demonstrated the applicability of the presented assay.