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%).     

Semi-automatic liquid chromatographic analysis of olpadronate in urine and serum using derivatization with (9-fluorenylmethyl)chloroformate

The semi-automatic bioanalytical assays for olpadronate [(3-dimethylamino-1-hydroxypropylidene)bisphosphonate] involves a protein precipitation with trichloroacetic acid and a double co-precipitation with calcium phosphate for serum samples and a triple calcium co-precipitation for urine samples. These manual procedures are followed by an automated solid-phase extraction on a cation-exchange phase. The procedure is continued either directly, at high olpadronate levels in urine, or after off-line evaporation under nitrogen and reconstitution in water on the same robotic workstation. The continued automatic procedure comprehends derivatization with (9-fluorenylmethyl)chloroformate, ion-pair liquid–liquid extraction and ion-pair HPLC with fluorescence detection at 274/307 nm. The intra- and inter-day precisions for urine and serum samples are typically in the 5–8% range for different olpadronate concentrations [levels near the lower limit of quantification (LLQ) excluded]. The LLQ is 5 ng/ml olpadronate for a 2.5-ml urine sample and 10 ng/ml for a 1-ml serum sample, respectively.     

9-Fluorenylmethoxycarbonylpyroglutamate, a side-product of derivatization of glutamate with 9-fluorenylmethyl chloroformate: a warning

9-Fluorenylmethyl chloroformate derivatization is widely used for determinations of amino acids in biological material. The derivatization of glutamate leads to the formation of 9-fluorenylmethoxycarbonylpyroglutamate as a side-product, which is thus a methodological artifact.     

Determination of polyamines in human tissues by precolumn derivatization with 9-fluorenylmethyl chloroformate and high-performance liquid chromatography

A high-performance liquid chromatography (HPLC) assay with fluorescence detection was developed for the determination of the polyamines putrescine, spermidine, spermine in samples of human spinal cord, cerebellum, cerebrospinal fluid (CSF), skeletal muscle, and muscle microdialysates without an extensive sample preparation. The precolumn derivatization was performed with 9-fluorenylmethyl chloroformate (FMOC), and the derivatizated polyamines were stable for at least 14 h at 4 degrees C. All polyamines were separated within 35 min. The method was checked for linearity, and mean correlation coefficient values of 0.995, 0.999, and 0.991 were achieved for putrescine, spermidine, and spermine, respectively. The within- and between-assay coefficient of variation percentages evaluated in standard solutions varied between 1.0 and 4.9% and between 1.3 and 6.9%, respectively. The corresponding values obtained in samples of human spinal cord were between 1.0 and 5.0% and between 0.6 and 5.8%. The values of the recovery, evaluated in spinal cord tissue, varied between 83.7 and 93.5%.     

Sensitive fluorimetric determination of gentamicin sulfate in biological matrices using solid-phase extraction, pre-column derivatization with 9-fluorenylmethyl chloroformate and reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic method is described for the determination of gentamicin in bacterial culture medium or plasma with increased sensitivity and improved separation of the C₁ component. Gentamicin was extracted from the biological matrix with high efficiency using carboxypropyl (CBA)-bonded silica. Derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) followed by C₁₈ reversed-phase chromatography allowed the fluorimetric detection of gentamicins C₁, C_1a and C₂. A fourth component, considered to be gentamicin C_2a, was partially resolved from the C₂ peak. Optimal conditions for the extraction and derivatization of gentamicin are described. The detection limit was below 50 μg/l, the assay was linear to 5 mg/1 and showed good reproducibility. It is concluded that pre-column derivatization with FMOC-Cl substantially improves the analysis of gentamicin compared with present methods based on reaction with o-phthaldialdehyde.     

Enantioselective determination of selfotel in human urine by high-performance liquid chromatography on a chiral stationary phase after derivatization with 9-fluorenylmethyl chloroformate

An analytical method for the enantioselective determination of selfotel in human urine has been developed and validated. The method is based on high-performance liquid chromatography and utilizes CGS 20005 (a selfotel analog) as the internal standard. Urine samples were derivatized in situ with o-phthalic dicarboxaldehyde–3-mercaptopropionic acid and 9-fluorenylmethyl chloroformate (FMOC). Chromatographic separations of the FMOC derivatives of selfotel enantiomers and the internal standard were achieved using a column switching system consisting of an Inertsil ODS-2 column (75×4.6 mm I.D., 5 μm) and a Chiralcel OD-R column (250×4.6 mm I.D., 10 μm). The composition of the mobile phase was acetonitrile–0.1 M phosphate buffer, pH 2.50 (35:65) for the Inertsil ODS-2 column and acetonitrile–0.1 M phosphate buffer, pH 2.00 (35:65) for the Chiralcel OD-R column. The analytes were monitored using fluorescence detection at an excitation wavelength of 262 nm and an emission wavelength of 314 nm. The limit of quantification (LOQ) for this method is 0.25 μg/ml for each selfotel enantiomer. The method was successfully utilized to determine preliminary selfotel stereospecific pharmacokinetics.     

Sensitive high-performance liquid chromatographic quantitation of gabapentin in human serum using liquid-liquid extraction and pre-column derivatization with 9-fluorenylmethyl chloroformate

Most of the published methods for analysis of gabapentin, an antiepileptic agent, in human serum are based on the same approach, involving o-phthaldialdehyde derivatization of deproteinized serum samples. The present paper however, describes a new, simple and sensitive high-performance liquid chromatographic method for determination of gabapentin in human serum using liquid-liquid extraction and 9-fluorenylmethyl chloroformate (FMOC-Cl) as pre-column labeling agent. The drug and an internal standard (azithromycin) were extracted from serum by salting-out approach using a mixture of dichloromethane-2 propanol (1:1, v/v) as the extracting solvent. The extracted analytes were subjected to derivatization with FMOC-Cl in the presence of phosphate buffer (pH 7). A mobile phase consisting of methanol-0.05 M sodium phosphate buffer (73/27, v/v; pH of 3.9) containing 1 ml/l triethylamine was eluted and chromatographic separation was performed on a Shimpack CLC-C18 (150 mm x 4.6 mm) column. The standard curve was linear over the range of 0.03-20 microg/ml and limit of quantification was 0.03 microg/ml. The performance of analysis was studied and the validated method showed excellent performance in terms of selectivity, specificity, sensitivity, precision and accuracy. No interferences were found from commonly co-administered antiepileptic agents.     

Amino acid analysis of collagen hydrolysates by reverse-phase high-performance liquid chromatography of 9-fluorenylmethyl chloroformate derivatives

A recently described procedure for amino acid analyses has been modified and adapted for use in quantitating the unique mixture of products commonly found in hydrolysates of the collagens. The method involves precolumn derivatization of hydrolysates with 9-fluorenylmethyl chloroformate (FMOC-CL), chromatographic separation of the derivatives and excess reagent on a reverse-phase column, and quantitation based on the fluorescent properties of the derivatives. The method takes advantage of the ease with which stable derivatives are formed with the FMOC reagent. Using a ternary gradient system, a complete amino acid analysis with good resolution of all components can be performed within 35 min. The sensitivity of the method is comparable to levels attained by other derivatives and the fluorescence response of each derivative is linear over the total range of 1-800 pmol. Given these parameters, the method allows complete amino acid analyses to be performed on 100 ng of collagen corresponding to a single picomole of a collagen chain (Mr 100,000).     

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.     

Gas chromatographic analysis of amino acid enantiomers in Carbetocin peptide hydrolysates after fast derivatization with pentafluoropropyl chloroformate

A novel sample preparation protocol for gas chromatographic (GC) analysis of amino acid enantiomers in peptides was developed. It comprises traditional acid hydrolysis, a novel treatment of the analytes with a fluoroalkyl chloroformate and GC/FID separation of enantiomers on a chiral capillary column. The major improvements consist in that the derivatization step proceeds in organic-aqueous media within seconds and the amino acid derivatives are volatile enough to suit the temperature range of the chiral Chirasil-Val capillary column. The approach was found beneficial for chiral analysis of pharmaceutically important Carbetocin peptide.     

High-sensitivity phenylthiohydantoin amino acid analysis using conventional and microbore chromatography

Reversed-phase microbore high-performance liquid chromatography was investigated for high-sensitivity analysis of phenylthiohydantoin (PTH) amino acids. A mixed nitrile alkylsilane bonded phase was developed and ternary gradient elution conditions were devised for resolution 150 × 4.6 mm I.D. column and transferred to a 150 × 1 mmI.D. microbore column. The performance of these columns was evaluated in terms of PTH amino acid resolution, enhanced sample detectability, and retention time precision. For this work a general purpose high-performance liquid chromatograph was modified to reduce extra column band broadening and a preformed gradient elution technique was developed to achieve rapid analysis times at microbore flow-rates. The microbore high-performance liquid chromatographic system is useful for high-sensitivity analysis of PTH amino acids in micro-sequencing applications.     

Quantitative determination of azithromycin in plasma, blood and isolated neutrophils by liquid chromatography using pre-column derivatization with 9-fluorenylmethyloxycarbonyl-chloride and fluorescence detection

In this study, a high-performance liquid chromatographic method with pre-column derivatization and fluorescence detection was optimised and validated for the quantification of azithromycin (AZM) in plasma. Clarithromycin (CLM) was used as an internal standard. Pre-column derivatization was done with 9-fluorenylmethyloxycarbonyl-chloride. Recovery from blood and polymorphonuclear neutrophils (PMNNs) isolated by a gravity separation procedure was also assessed. Analytical separation was carried out using a C18 column as stationary phase and acetonitril-phosphatebuffer as mobile phase. Peak quantification was carried out by excitation at 26 7 nm and detection at 317 nm. A lower limit of quantitation of 0.042+/-0.017 mg/l in plasma, 0.119+/-0.065 mg/l in blood and 0.072+/-0.036 in water was achieved. Linearity was assessed from 0 to 1.5mg/l in plasma and blood and from 0-9 mg/l in water. The analytical method proved to be applicable in a pharmacokinetic study of AZM in a Cystic Fibrosis patient.     

High-sensitivity amino acid analysis: methodology for the determination of amino acid compositions with less than 100 picomoles of peptides

Highly sensitive methodology is described for the determination of amino acid compositions of picomole quantities of peptides using an automated fluorescence amino acid analyzer with o-phthalaldehyde as the detection agent. All commonly occurring amino acids, including cystine, proline, and tryptophan, can be quantitated. High sensitivity is primarily achieved by using simple procedures which effectively and reliably reduce the level of interfering contamination present in buffers and reagents or introduced during sample handling. Accurate and reproducible amino acid compositions are obtained with 50 pmol or less peptide.     

A simple method for the assay of colistin in human plasma, using pre-column derivatization with 9-fluorenylmethyl chloroformate in solid-phase extraction cartridges and reversed-phase high-performance liquid chromatography

A simple, selective and sensitive high-performance liquid chromatographic (HPLC) method is described for the determination of colistin in human plasma. Derivatization with 9-fluorenylmethyl chloroformate was performed in the same solid-phase extraction C₁₈ cartridge used for sample pre-treatment, followed by reversed-phase HPLC with fluorimetric detection. Quantification was achieved using the ratio of the summed peak areas of colistin A and B derivatives to that of the derivative of netilmicin (internal standard). Linear calibration curves were obtained within the concentrations of colistin sulfate from 0.10 to 4.0 mg/l in plasma. Accuracy was within 10% and reproducibility (RSD) was less than 10%.     

9-fluorenylmethyl chloroformate as a fluorescence-labeling reagent for derivatization of carboxylic acid moiety of sodium valproate using liquid chromatography/tandem mass spectrometry for binding characterization: a human pharmacokinetic study

In High Performance Liquid Chromatographic (HPLC) determination of chemicals with acidic functions, different labeling agents are used to improve sensitivity of the assay. 9-Fluorenylmethyl chloroformate (FMOC-Cl), on the other hand, is a suitable labeling agent, which reacts with both primary and secondary amines and less readily with hydroxyl groups in alkaline conditions. However, the reagent has not been applied in labeling of chemicals with acidic function yet. In this study which is the first report on application of FMOC-Cl in derivatization and analysis of a drug with acidic function, valproic acid (VPA), one of a series of fatty carboxylic acids with anticonvulsant activity, was derivatized using the reagent and quantified in serum samples by HPLC with fluorescence detection. In addition, to document the reaction between the labeling agent and carboxylic acid moiety of the drug, we developed a liquid chromatography-tandem MS/MS (LC-MS/MS) method. Following liquid-liquid extraction, derivatization of the drug and an internal standard was achieved in alkaline medium. The elute was monitored by a fluorescence detector with respective excitation and emission wavelengths of 265 and 315 nm. The present method is more sensitive comparing with other published HPLC procedures for analysis of VPA. The assay is sensitive enough to measure drug levels obtained in human single dose studies with a limit of quantification of 0.01 μg/mL. Also the method is linear over the concentrations range of 0.01-32 μg/mL of VPA in human serum using 100 μL serum sample and 5 μL injection. The coefficient variation values of both inter and intra day analysis were less than 12% and the percentage error was less than 4%. The method performance was studied and the validated procedure applied in a randomized cross-over bioequivalence study of two different VPA preparations in 24 healthy volunteers.     

Separation and quantitative analysis of O-linked phosphoamino acids by isocratic high-performance liquid chromatography of the 9-fluorenylmethyl chloroformate derivatives

Phosphoamino acids derivatized with 9-fluorenylmethyl chloroformate were separated on an anion-exchange column (Partisil 10 SAX) at pH 3.90 using an isocratic elution with 10.0 mM potassium phosphate, 1.0% tetrahydrofuran, and 55% methanol. Phosphoamino acids were eluted with baseline resolution in the following order: phosphotyrosine, phosphothreonine, and phosphoserine. Each phosphoamino acid was separated from its parent amino acid, dicarboxylic amino acids, sugaramine phosphates, as well as the other common amino acids. The turn-around time from injection to injection was 35 min. The linearity for all three O-linked phosphoamino acids extended from 0.5-1000 pmol and has been shown to be directly applicable to the analysis of isolated phosphoproteins.     

New approach for amino acid profiling in human plasma by selective fluorescence derivatization

A new approach for the separation of 6-aminoquinolyl-carbamyl (AQC)-derivatized amino acids has been proposed. The chromatography used ion-pairing mechanism to increase the method selectivity. Mobile phase was based on triethylamine buffer containing N,N-dimethyloctylamine as a modifier. A number of factors, buffer composition and pH, counterion concentration, temperature and acetonitrile gradient profile, were optimized to achieve final chromatographic conditions. With the presented analytical method, the separation and identification of 34 AQC-amino acids and amino compounds present in human plasma is possible. The results of validation proved the applicability of the method for quantification of 27 amino acids in biological samples. The ultrafiltration proposed as deproteinization procedure gave repeatable and reliable results for the amino acids under investigation. This method introduced in routine testing can be a suitable tool for amino acid profiling in plasma including all aspects of clinical application.     

Simultaneous determination of vigabatrin and amino acid neurotransmitters in brain microdialysates by capillary electrophoresis with laser-induced fluorescence detection

Capillary electrophoresis with laser-induced fluorescence detection (CE-LIFD) coupled to in vivo microdialysis sampling was used in order to monitor simultaneously a drug and several neurotransmitters in the brain extracellular fluid. Determination of the antiepileptic drug vigabatrin and the amino acid neurotransmitters glutamate (Glu), l-aspartate (l-Asp) and gamma-aminobutyric acid (GABA) was performed on low-concentration samples which were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) and separated using a pH 9.2 75 mM sodium borate running buffer containing 60 mM sodium dodecyl sulfate (SDS) and 5mM hydroxypropyl-beta-cyclodextrin (HP-beta-CD). Glu, l-Asp and vigabatrin derivatized at a concentration of 1.0 x 10(-9) M, and GABA derivatized at a concentration of 5.0 x 10(-9) M, produced peaks with signal-to-noise ratios of 8:1, 8:1, 4:1 and 5:1, respectively. The nature of the neurotransmitter peaks found in rat brain microdialysates was confirmed by both electrophoretic and pharmacological validations. This method was used for monitoring vigabatrin and amino acid neurotransmitters in microdialysates from the rat striatum during intracerebral infusion of the drug and revealed rapid vigabatrin-induced changes in GABA and Glu levels. This original application of CE-LIFD coupled to microdialysis represents a powerful tool for pharmacokinetic/pharmacodynamic investigations.     

Single-step enantioselective amino acid flux analysis by capillary electrophoresis using on-line sample preconcentration with chemical derivatization

Capillary electrophoresis (CE) represents a versatile platform for integrating sample pretreatment with chemical analysis because of its ability to tune analyte electromigration and band dispersion properties in discontinuous electrolyte systems. In this article, a single-step method that combines on-line sample preconcentration with in-capillary chemical derivatization is developed for rapid, sensitive, and enantioselective analysis of micromolar levels of amino acids that lack intrinsic chromophores by CE with UV detection. Time-resolved electrophoretic studies revealed two distinct stages of amino acid band narrowing within the original long sample injection plug occurring both prior to and after in-capillary labeling via zone passing by ortho-phthalaldehyde/N-acetyl l-cysteine (OPA/NAC). This technique enabled direct analysis of d-amino acids in a 95% enantiomeric excess mixture with sub-micromolar detection limits and minimal sample handling, where the capillary functions as a preconcentrator, microreactor, and chiral selector. On-line sample preconcentration with chemical derivatization CE (SPCD-CE) was applied to study the enantioselective amino acid flux in Escherichia coli bacteria cultures, which demonstrated a unique l-Ala efflux into the extracellular medium. New strategies for high-throughput analyses of low-abundance metabolites are important for understanding fundamental physiological processes in bacteria required for screening the efficacy of new classes of antibiotics as well as altered metabolism in genetically modified mutant strains.     

Determination of 9-nitrocamptothecin by precolumn derivatization and its metabolite 9-aminocamptothecin in a biological fluid using reversed-phase high-performance liquid chromatography with fluorescence detection

A novel insoluble topoisomerase I inhibitor, 9-nitrocamptothecin (9-NC), is in advanced stages of clinical development and has been used to treat a diverse array of tumor types, including breast, ovarian, pancreatic and haematological malignancies. We have established a sensitive high-performance liquid chromatography method using fluorescence detection for the quantitation of 9-NC. Non-fluorescent 9-NC is converted to fluorescent 9-aminocamptothecin (9-AC) via a one-step pre-column derivative reaction. The quantitative limit of 9-NC was 1 ng/ml and the method was reproducible with the respective intra- and inter-day variability falling below 5.0 and 9.0%. The determination of both 9-NC and its metabolite 9-AC in dog plasma was also achieved using the same chromatographic and detection conditions. In dog plasma, the quantitative limits of 9-AC and 9-NC were 0.25 and 1 ng/ml, respectively. The presence of 9-AC in the samples yielded no interference with the determination of 9-NC. However, individual matrices can affect the conversion efficiency of 9-NC, thus indicating that standard samples should be run for each matrix.