G. Ali Qureshi

Application of high-performance liquid chromatography to the determination of free amino acids in physiological fluids

Multiple-step gradient systems were used for the analysis of free amino acids in physiological fluids by high-performance liquid chromatography with fluorescence detection on two reversed-phase C18 columns. Standard amino acids, plasma or urine samples were subjected to derivatization with ophthalaldehyde in the presence of mercaptoethanol before the separation was performed. More than 22 amino acids were separated in less than 1 h on either 5-micron Ultrasphere-ODS columns or 5-micron Resolve C18 columns by using a two-solvent system. The correlation of the integrated peak areas with the concentration of all amino acids showed a linear relationship between 10 and 150 pmol per 20-microliter injection for all The method has a lower detection limit of less than 1 pmol per 20-microliter injection for all amino acids. Quantitative analysis of micro amounts of amino acids in plasma and urine by the internal standard method gave highly reproducible results with a mean coefficient of variation of less than 3% and r2 = 0.999. Because of the simplicity of the method its application provides a better means for closely monitoring the patients undergoing dialysis and treatment for renal disorders. These results are compared with those from classical ion-exchange chromatography.

Determination of clenbuterol and mabuterol in equine plasma by ion-pair liquid chromatography with electrochemical detection : Chromatographic and electrochemical characteristics

A method for the routine determination of the beta-adrenergic drugs clenbuterol and mabuterol in equine plasma has been developed. The drugs were isolated from alkalinized plasma by liquid-liquid extraction. The organic phase was evaporated to dryness and the residue was dissolved in the mobile phase prior to injection. The recoveries were 98% and 95% for clenbuterol and mabuterol, respectively. The drugs were separated by reversed-phase high-performance liquid chromatography and quantitated by a use of a coulometric detector set at +0.75 V vs. the internal reference electrode. The influence of pH and amounts of organic modifier and ion-pairing agent on the retention times was investigated. The relationship between peak current and concentration was linear up to 1 microgram/ml for both compounds. The limits of detection were 0.5 ng/ml for clenbuterol and 2 ng/ml for mabuterol with a signal-to-noise ratio of 3. A brief discussion of the electrochemistry of the compounds is given.

Determination of free amino acids in biological samples: Problems of quantitation

An automatic on-line high-performance liquid chromatographic method was developed to study the effects of various precipitating agents and delayed deproteinization procedures on the estimation of plasma levels of amino acids. The optimized method for analysis is based on pre-column derivatization with o-phthalaldehyde in the presence of 2-mercaptoethanol. The separation of 25 amino acids is accomplished within 45 min on a 5-microns C18 column, using a multi-step gradient with two solvents. The method is sensitive and reproducible, and the relationship between the fluorescence intensity and concentration is linear for each amino acid over a wide concentration range.

Quantitation of free amino acids in biological samples by high-performance liquid chromatography : Application of the method in evaluating amino acid levels in cerebrospinal fluid and plasma of patients with multiple sclerosis

An automatic on-line high-performance liquid chromatographic method based on a precolumn derivatization with o-phthalaldehyde has been developed to quantitate levels of free amino acids in cerebrospinal fluid (CSF) and plasma samples from 12 patients with multiple sclerosis (MS) and 12 controls. The analytical method gave reproducible results with relative standard deviations of 0.5-3% for all amino acids. The separation of 24 amino acids was performed on a reversed-phase C18 column, using two solvents and a multiple-step gradient. Each chromatographic experiment was completed within 40 min. The results showed higher levels of Glu, Gln, Gly and Ala and lower levels of Met, Val, Phe and Lys in plasma of MS patients. In CSF, increased levels of Gln, Arg, Ser and Tyr and decreased levels of Asp, Glu, Met, gamma-aminobutyric acid and Phe were found in MS patients, whereas the levels of other amino acids remained more or the less same in both groups.

Is the Deficiency of Vitamin B12 Related to Oxidative Stress and Neurotoxicity in Parkinsons Patients

This review deals with the results showing the relation between vitamin B(12) deficiency and neurotoxicity of homocysteine and nitrite (a metabolite of nitric oxide) in Parkinsons patients treated with levodopa (L-Dopa). We have already reported a linear relationship between the CSF levels of nitrite with glutamic acid and homocysteine suggesting that the production of nitrite is interrelated with the neurotoxic level of homocysteine. The levels of nitrite and homocysteine resulting in the deficiency of vitamin B(12) are some of the factors promoting degeneration in Parkinsons disease. This review emphasizes the importance of these parameters in designing suitable drug therapy for Parkinson disease. Additionally, there is evidence that increased homocysteine levels might accelerate dopaminergic cell death in Parkinson disease (PD), through neurotoxic effects. Furthermore, levodopa (L-Dopa) treatment of PD results in hyperhomocysteinemia as a consequence of L-Dopa methylation by catechol-O-methyltransferase (COMT). Therefore, higher dietary intakes of folate, vitamin B12, and vitamin B6 might decrease the risk of PD through decreasing plasma homocysteine.

High-performance liquid chromatographic methods with fluorescence detection for the determination of branched-chain amino acids and their alpha-keto analogues in plasma samples of healthy subjects and uraemic patients

High-performance liquid chromatographic (HPLC) methods have been developed for the quantification of branched-chain amino acids (BCAA) and their keto analogues (BCKA). Amino acids and their keto analogues were derivatized with o-phthalaldehyde, 2-mercaptoethanol and o-phenylenediamine sulphate prior to HPLC. Both separations were performed on a reversed-phase column, using a multi-step gradient system with two solvents and a fluorescence detector. These methods are simple and sensitive and give highly reproducible results. By using an automatic system, the instability problem is avoided and the reaction kinetics are controlled. The use of a simple clean-up procedure with preparative cation-exchange chromatography before BCKA analysis concentrates the dilute plasma sample. The methods were applied to the determination of BCAA and BCKA in plasma samples of healthy volunteers and patients with chronic renal disorders. The relationship between the concentrations of BCAA and BCKA in plasma for these two groups is shown.

The emerging role of iron, zinc, copper, magnesium and selenium and oxidative stress in health and diseases

The role of various metals in health and disease of specific pathological conditions is well-documented; however, the role, interaction and consequences of deficiency and toxicity of any specific metal in many diseases at one time has not yet been described fully. In this review, the role of iron, zinc, copper, magnesium and selenium is shown in various pathological conditions. The purpose is to find the missing link between any one metal and various diseases so that conclusions may be drawn based on the consequences of the metal deficiency and toxicity.

Determination of histidine and 3-methylhistidine in physiological fluids by high-performance liquid chromatography.

High-performance liquid chromatography based on pre-column derivatization of histidine and 3-methylhistidine with o-phthalaldehyde-mercaptoethanol has been used to determine these amino acids in small volumes of plasma and urine. The elution is performed in 45 min on a 5-micron Resolve C18 column by a multi-step gradient. The eluted analytes are measured with a fluorescence detector which provides detection limits of less than 1 pmol per 20-microliter injection. The correlation between concentration and the integrated area of amino acids gives a linear relationship between 10 and 150 pmol per injection. Some preliminary results from patients with chronic renal failure under variation of diet are presented.

Quantitation of free amino acids in plasma and muscle samples in healthy subjects and uremic patients by high-performance liquid chromatography and fluorescence detection

An automatic on-line high-performance liquid chromatographic (HPLC) method is developed to quantitate free amino acids in the biological samples. The method is based on pre-column derivatization of amino acids with orthophthalaldehyde (OPA) in presence of 2-mercaptoethanol (2-ME). The derivatized amino acids were separated on a 5 micron C18 column using a multi-step gradient with two solvents and the detection was made at Eex = 340 nm and Eem = 450 nm. The results were highly reproducible with a relative standard deviation (RSD) between 0.5-2% for all amino acids. Each chromatographic run was completed within 40 min to separate 24 amino acids. The optimized method was applied to evaluate the levels of free amino acids in plasma and muscle samples of eight healthy subjects and 13 uremic patients under fasting conditions.

Cholecystokinin peptides in cerebrospinal fluid : a study in healthy male subjects

The clinical reliability of measuring cholecystokinin (CCK) peptides in the cerebrospinal fluid (CSF) has not been fully elucidated. Therefore, we have assayed CCK-8S and CCK-4 in CSF obtained from 14 healthy male subjects, lumbar-punctured at the L4-5 level following a strictly standardised procedure. CSF concentrations of free CCK-8S and free CCK-4 were used as dependent variables while age, height, body weight, atmospheric pressure and some other factors served as independent variables. It was shown that the CCK-8S ratio between the second (7-12 ml) and first (0-6 ml) CSF fractions, correlated significantly with the atmosphere pressure at the time of puncture. Neither CCK-8S nor CCK-4 displayed concentration gradients in CSF. The CCK-4 levels, expressed as pmol l-1 in the total amount of CSF were found to be positively correlated with the neuraxis distance in the lying position and negatively with the neuraxis distance in the sitting position. Furthermore, CCK-4, expressed as pmol l-1 per min of tapping-time (pmol l-1 min-1), showed a negative correlation with storage time, presumably mirroring a proteolytic process. CCK-8S and CCK-4 intercorrelated positively independently of whether expressed as pmol l-1 or pmol l-1 min-1. In conclusion, the results of this exploratory study indicate that the neuraxis distance (in the sitting and lying positions) and storage-time have to be accounted for when interpreting data on CSF levels of CCK-4. Attention has to be paid to the potential influence of atmospheric pressure on the concentration ratio of CCK-8S.