Stereoselective RP-HPLC determination of esmolol enantiomers in human plasma after pre-column derivatization

A stereoselective reversed-phase HPLC assay to determine S-(-) and R-(+) enantiomers of esmolol in human plasma was developed. The method involved liquid-liquid extraction of esmolol from human plasma, using S-(-)-propranolol as the internal standard, and employed 2,3,4,6-tetra-O-acetyl-beta-d-glucopyranosyl isothiocyanate as a pre-column chiral derivatization reagent. The derivatized products were separated on a 5-microm reversed-phase C18 column with a mixture of acetonitrile/0.02 mol/L phosphate buffer (pH 4.5) (55:45, v/v) as mobile phase. The detection of esmolol derivatives was made at lambda=224 nm with UV detector. The assay was linear from 0.035 to 12 microg/ml for each enantiomer. The analytical method afforded average recoveries of 94.8% and 95.5% for S-(-)- and R-(+)-esmolol, respectively. For each enantiomer, the limit of detection was 0.003 microg/ml and the limit of quantification for the method was 0.035 microg/ml (RSD<14%). The reproducibility of the assay was satisfactory.     

Liquid chromatographic determination of sodium mercaptoundecahydrododecaborate in rat urine and plasma after precolumn derivatization

A high-performance liquid chromatographic (HPLC) method was developed for the determination of disodium mercaptoundecahydrododecaborate (BSH) in biological fluids. Monobromobimane was used as a precolumn derivatizing agent. A stable derivative was obtained. The derivative was separated on a C₁₈ column using reversed-phase ion-pairing chromatography and detected by a spectrophotometric detector at 373 nm. The detection limit was 200 ng/ml (0.1 ppm boron). Calibration curves were prepared for rat urine and plasma samples. The calibration curves were linear in the range of 1 μg/ml to 100 μg/ml for urine samples and 0.2 μg/ml to 50 μg/ml for plasma samples.     

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.     

Liquid chromatographic determination of amodiaquine in human plasma

A normal-phase high-performance liquid chromatographic method using dichloromethane- methanol-1M perchloric acid (100:10:0.9, v/v/v) at a flow rate of 1.0 ml min(-1) on a LiChrospher Si column with UV (254 nm) detection has been developed for the determination of amodiaquine and its metabolites desethyl amodiaquine and bisdesethyl amodiaquine in plasma. The limit of quantification was 5 ng ml(-1). Mean within-day and day-to-day coefficients of variation (CV) were 4.10 and 6.27% for amodiaquine, 3.43 and 4.80% for desethyl amodiaquine and 3.53 and 5.23% for bisdesethyl amodiaquine, respectively. Mean extraction recovery of amodiaquine, desethyl amodiaquine and bisdesethyl amodiaquine from plasma were 82.48, 74.50 and 69.65%, respectively. Chloroquine and its metabolite desethyl chloroquine, quinine, sulfadoxine and primaquine do not interfere in the detection of amodiaquine, desethyl amodiaquine and bisdesethyl amodiaquine in plasma.     

Liquid chromatographic determination of irbesartan in human plasma

A simple and sensitive method was developed for determination of irbesartan by liquid chromatography with fluorescence detection. Irbesartan and losartan (I.S.) in human plasma were extracted using diethyl ether:dichloromethane (7:3, v/v) followed by back extraction with 0.05 M sodium hydroxide. Neutralized samples were analyzed using 0.01 M potassium dihydrogen phosphate buffer (containing 0.07% triethylamine as peak modifier, pH was adjusted with orthophosphoric acid to pH 3.0) and acetonitrile (66:34, v/v). Chromatographic separation was achieved on an ODS-C-18 column (100 mm x 4.6 mm i.d., particle size 5 microm) using isocratic elution (at flow rate 1.25 ml/min). The peak was detected using a fluorescence detector set at Ex 259 nm and Em 385 nm, and the total time for a chromatographic separation was approximately 13 min. The validated quantitation ranges of this method were 15-4000 ng/ml with coefficients of variation between 0.75 and 12.53%. Mean recoveries were 73.3-77.1% with coefficients of variation of 3.7-6.3%. The between- and within-batch precision were 0.4-2.2% and 0.9-6.2%, respectively. The between- and within-batch relative errors (bias) were (-5.5) to 0.9% and (-0.6) to 6.9%, respectively. Stability of irbesartan in plasma was >89%, with no evidence of degradation during sample processing and 60 days storage in a deep freezer at -70 degrees C. This validated method is sensitive and simple with between-batch precision of <3% and can be used for pharmacokinetic studies.     

Sensitive determination of trimetazidine in spiked human plasma by HPLC with fluorescence detection after pre-column derivatization with 9-fluorenylmethyl chloroformate

A high-performance liquid chromatographic method for the determination of trimetazidine dihydrochloride (TMZ) in spiked human plasma is described. The method is based on the pre-column derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) using the fluorimetric detection technique. Fluoxetine HCl (FLX) was used as internal standard. Both, TMZ and FLX were completely derivatized after heating at 50 degrees C for 20 min in borate buffer pH 8.0. Samples were analyzed by high performance liquid chromatography (HPLC) using Zorbax-TMS column (250 mm x 4.6 mm, i.d., 5 microm) and mobile phase consist of acetonitrile, methanol and 20 mM sodium acetate pH 4.7 (44:6:50; v/v/v). Fluorescence detector (FLD) was adjusted at excitation and emission wavelengths; 265 and 311 nm, respectively. The linearity of the method was in the range of 4.5-200 ng/ml. Limits of detection (LOD) and quantification (LOQ) were 1.5 and 4.5 ng/ml, respectively. Trimetazidine recovery was 96.5+/-1.3% (n=6; RSD=2.1%).     

Analysis of polyamines in biological samples by HPLC involving pre-column derivatization with o-phthalaldehyde and N-acetyl-l-cysteine

Polyamines (putrescine, spermine and spermidine) play a crucial role in the regulation of cell growth, differentiation, death and function. Accurate measurement of these substances is essential for studying their metabolism in cells. This protocol describes detailed procedures for sample preparation and HPLC analysis of polyamines and related molecules (e.g., agmatine and cadaverine) in biological samples. The method is optimized for the deproteinization of samples, including biological fluids (e.g., 10 μl), plant and animal tissues (e.g., 50 mg), and isolated/cultured cells (e.g., 1 × 10⁶ cells). The in-line reaction of polyamines with o-phthalaldehyde and N-acetyl-l-cysteine yields fluorescent derivatives which are separated on a reversed-phase C₁₈ column and detected by a fluorometer at an excitation wavelength of 340 nm and an emission wavelength of 450 nm. The total running time for each sample (including column regeneration on the automated system) is 30 min. The detection limit is 0.5 nmol/ml or 0.1 nmol/mg tissue in biological samples. The assays are linear between 1 and 50 μM for each of the polyamines. The accuracy (the nearness of an experimental value to the true value) and precision (agreement between replicate measurement) of the HPLC method are 2.5–4.2 % and 0.5–1.4 %, respectively, for biological samples, depending on polyamine concentrations and sample type. Our HPLC method is highly sensitive, specific, accurate, easily automated, and capable for the analysis of samples with different characteristics and small volume/amount, and provides a useful research tool for studying the biochemistry, physiology, and pharmacology of polyamines and related substances.     

Quantitative high-performance liquid chromatographic determination of acrolein in plasma after derivatization with Luminarin 3

A rapid, sensitive and specific high-performance liquid chromatographic method for the quantification of acrolein (1), one of the toxic metabolites of oxazaphosphorine alkylating agents (cyclophosphamide and ifosfamide) was developed. Condensation of acrolein with Luminarin^® 3 afforded a fluorescent derivative that could be specifically detected and quantified. Chromatographic conditions involved a C₁₈ RP column Uptisphere and a gradient elution system to optimize resolution and time analysis. The method showed high sensitivity with a limit of detection of 100 pmol/ml and a limit of quantification of 300 pmol/ml. This technique is particularly suitable for pharmacokinetic studies on plasma of oxazaphosphorine-receiving patients.     

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%.     

High-performance liquid chromatographic determination of histamine in biological samples after derivation with o-phthalaldehyde

A high-performance liquid chromatographic (HPLC) method for the determination of histamine in tissues, based on precolumn derivation with o-phthalaldehyde, is described. Trichloroacetic acid extracts of rat brain, but not of rat stomach or of rat peritoneal mast cells, had to be cleaned-up by a chromatographic step before HPLC. The extracts were allowed to react with o-phthalaldehyde at pH 12.5 and -20 degrees C for 12 h, followed by acidification to pH 2.0. HPLC was performed on a reverse-phase column with isocratic elution using sulfuric acid in methanol as solvent system. A fluorescence detection system was used; excitation was set at 353 nm and emission was read at 451 nm. One chromatographic run was completed in 20 min. The detection limit with the conventional procedure was 1.5 ng histamine per sample, with a scaled-down procedure it was 250 pg per sample. With extracts of rat gastric mucosa the within-run variation was 2.7% and the day-to-day variation 4.5%.     

Liquid chromatographic method for the determination of rosiglitazone in human plasma

A robust, accurate and sensitive high-performance liquid chromatographic method for the determination of rosiglitazone (I) in human plasma has been developed. Pioglitazone (II) was used as internal standard. Both I and II are extracted from plasma using a liquid-liquid extraction procedure. Isocratic separation of I and II is carried out using a reversed-phase Zorbax SB C(18), 15-cm column with mobile phase consisting of methanol and a mixed phosphate buffer (10 mM monobasic sodium phosphate and dibasic sodium phosphate, pH adjusted to 2.6 with ortho-phosphoric acid) in the ratio 30:70 (v/v) and quantified by UV detection at 245 nm. Linearity was established over the range 5-1250 ng/ml using 1 ml human plasma. The method is specific, the endogenous components in plasma do not interfere with I and II. C.V. (%) of intra-day samples is less than 5.0% at four concentrations tested namely 5, 10, 500 and 1000 ng/ml. Similarly, over the same nominal concentrations, the precision of inter-day (5 days) samples also results in C.V. (%) less than 5.0%. The recoveries of I and II from human plasma were about 79 and 60%, respectively. This method can be used for routine clinical monitoring of I.     

Liquid chromatographic method for the determination of ticlopidine in human plasma

A simple high-performance liquid chromatographic method for determination of ticlopidine in human plasma using ultra violet detection was developed. The separation of the investigated compound and internal standard was achieved on a C₁₈ BD column with a 0.01 M potassium dihydrogen phosphate buffer (pH 4)–acetonitrile–methanol (20:40:40, v/v) mobile phase. The detection was performed at 215 nm. The compounds were isolated from plasma by Bond Elut C₁₈ solid-phase extraction, the mean absolute recovery was 84.9%. The limit of quantitation was 10 ng ml⁻¹, the limit of detection was 5 ng ml⁻¹. The bioanalytical method was validated with respect to linearity, within- and between-day accuracy and precision, system suitability and stability. All validated parameters were found to be within the internationally required limits. The developed analytical method for ticlopidine was found to be suitable for application in pharmacokinetic studies and human drug monitoring.     

Liquid chromatographic method for the determination of rizatriptan in human plasma

A high-performance liquid chromatographic (HPLC) method with fluorescence detection has been developed for the determination of rizatriptan in human plasma. Following a single-step liquid-liquid extraction with methyl tertiarybutyl ether, the analytes were separated using a mobile phase consisting of 0.05% (v/v) triethylamine in water (adjusting to pH 2.75 with 85% phosphoric acid) and acetonitrile (92:8, v/v). Fluorescence detection was performed at an excitation wavelength of 225nm and an emission wavelength of 360nm. The linearity for rizatriptan was within the concentration range of 0.5-50ng/ml. The intra- and inter-day precisions of the method were not more than 8.0%. The lower limit of quantification (LLOQ) was 0.5ng/ml for rizatriptan. The method was sensitive, simple and repeatable enough to be used in pharmacokinetic studies.     

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.     

Liquid chromatographic determination of triethylenetetramine in human and rabbit sera based on intramolecular excimer-forming fluorescence derivatization

A highly selective and simple fluorimetric liquid chromatographic method for the determination of triethylenetetramine (TETA), a therapeutic drug for Wilson's disease, in human and rabbit sera is described. This method is based on intramolecular excimer-forming fluorescence derivatization, which allows spectrofluorometric discrimination of polyamino compounds from monoamino species, followed by liquid chromatography. TETA and 1,6-hexanediamine (internal standard) were converted to the corresponding excimer-forming derivatives with a pyrene reagent, 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester. The derivatives were separated within 20 min on a reversed-phase column using isocratic elution and detected spectofluorometrically at 480 nm with excitation at 345 nm. This method was successfully applied to the monitoring of TETA in human and rabbit sera with a simple pretreatment. The detection limit for TETA in serum was 18 ng/ml (0.13 nmol/ml) corresponding to 0.2 pmol on column at a signal-to-noise ratio of 3.     

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.     

Liquid chromatography--mass spectrometric method combined with derivatization for determination of 1 alpha-hydroxyvitamin D(3) in human plasma

A stable isotope dilution liquid chromatography-tandem mass spectrometric (LC-MS-MS) method for the determination of plasma 1 alpha-hydroxyvitamin D(3) [1 alpha(OH)D(3)] has been developed. The method employed derivatization, the reaction with 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione and acetylation, which significantly improved the ionization efficiency of 1 alpha(OH)D(3) with a detection limit of 6.3 fmol per injection. The plasma 1 alpha(OH)D(3) was extracted with acetonitrile, purified with disposable cartridges, derivatized and subjected to LC-MS-MS analysis using atmospheric pressure chemical ionization. The intra- and inter-assay coefficients of variation were below 10.6 and 4.7%, respectively, and the analytical recovery of 1 alpha(OH)D(3) was quantitative. The limit of quantitation was 25 pg/ml for a 1.0-ml plasma aliquot. The application of the developed method to the sample of a volunteer orally given 1 alpha(OH)D(3) was also described.     

Semi-automatic liquid chromatographic analysis of pamidronate in serum and citrate plasma after derivatization with 1-naphthylisothiocyanate

The semi-automatic method for the determination of the bisphosphonate pamidronate in serum and citrate plasma involves a manual protein precipitation with trichloroacetic acid and a manual coprecipitation of the bisphosphonate with calcium phosphate, followed by an automated solid-phase extraction on anion-exchange columns. After off-line evaporation of the extract under nitrogen and reconstitution in water, the automatic procedure is continued by automatic derivatization with 1-naphthylisothiocyanate, ion-pair liquid–liquid extraction and a treatment with hydrogen peroxide, prior to analysis by ion-pair HPLC and fluorescence detection at 285/390 nm. The intra- and inter-day precisions are 1.3 and 7%, respectively, for a standard of 100 ng ml⁻¹ pamidronate in serum; the average accuracy for this standard is 107%. The lower limit of quantification is 20 ng ml⁻¹ pamidronate in 1 ml of human serum.     

High-performance liquid chromatographic analysis of physiological amino acids in human brain tumors by pre-column derivatization with phenylisothiocyanate

A reversed-phase high-performance liquid chromatographic technique for the determination of free amino acids in five biopsies of human brain tumors (two meningiomas, one glioblastoma and two oligodendrogliomas) is described. The frozen tissues were homogenized, deproteinized with perchloric acid and neutralized with potassium hydroxide. Aliquots of the supernatant containing the physiological amino acids are used for pre-column derivatization with phenylisothiocyanate. The derivatized PTC-amino acids (phenylthiocarbamyl derivatives) are stable for a five day period if stored as a powder at −20°C in an inert atmosphere and they can be analyzed on a reversed-phase column (PicoTag) using a gradient of two eluents with absorption detection at a wavelength of 254 nm. Good resolution of several amino acids (>30) is achieved within ca. 60 min. For most amino acids this method is suitable for an accurate measurement over a wide range of physiological concentrations (50–400 pmol) starting from a very small amount of sample.     

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.