Reversed-phase High-performance Liquid Chromatographic Procedure Research Articles

A single liquid chromatography procedure to concentrate, separate and collect size-selected polyynes produced by pulsed laser ablation in water

Polyynes are linear carbon chains characterized by alternated single and triple bonds and terminated by hydrogen or other terminal substituents. They can be synthesized by pulsed laser ablation in liquid (PLAL) as a scalable, cost-effective, and fast physical technique. Water can be employed as a solvent for PLAL to avoid toxicity problems and to reduce costs compared to organic solvents. However, in in this case, the production yield of polyynes reached is extremely low and prevents further characterization and implementation in new functional materials. In this work, we synthesized polyynes by pulsed laser ablation in water and we optimized the process parameters to improve the yield of polyynes by PLAL. Then, we developed a procedure entirely based on reversed-phase high-performance liquid chromatography (RP-HPLC) which effectively enables the concentration, separation and collection of polyynes according to their length. Since the polyynes sample is an aqueous solution, we could inject it directly into the RP-HPLC column without the dilution step required in the case of a sample in an organic solvent. Thanks to our single RP-HPLC procedure, it is possible to highly increase the concentration and separately characterize different size-selected polyynes for further use in functional materials.

SIMULTANEOUS ESTIMATION OF CLONAZEPAM AND METRONIDAZOLE IN PHARMACEUTICAL TABLETS BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY MODE WITH UV DETECTION

Clonazepam (CLO) plays a significant role in treating seizures, myoclonic seizures, and other clinical infections, while metronidazole (MTZ) is an antiprotozoal, antibacterial drug. The reversed-phase high performance liquid chromatographic (RP-HPLC) technique has provided greater precision and sensitivity over other methods, especially the colorimetric and spectrophotometric. This study aimed to implement a simple method in pure and pharmaceutical tablets for the simultaneous determination of CLO and MTZ. The RPHPLC method's development and optimization for validating a separation method for the simultaneous estimation of both drugs in pharmaceutical formulation involved studying the optimum mobile phase, buffer concentration, and the pH value. Under the chromatographic conditions, RP-HPLC system achieved excellent separation on a Phenomenex HyperClone BDS (250 x 4.60 mm, 130A, and 5μ) at a temperature of 45°C and the following conditions: 65:35% acetonitrile:acetic acid and sodium acetate buffer solution (NaOAc/HAc) as mobile phase (pH 3.5); 20 μL injection volume at a flow rate of 0.75 mL/min and detection wavelength of 310 nm. The proposed RP-HPLC procedure demonstrated high precision (RSD% 1%), as well as the good linear relationship of the calibration graph at concentration ranges of 50-160, 35-100 ppb with a coefficient of determination (r2) of the regression line of 0.9996 and 0.9997 for metronidazole and clonazepam respectively. The proposed method offered excellent validated values for both LOD (4.24 and 3.06 ppb) and LOQ (14.15– 10.21 ppb) for both metronidazole and clonazepam drugs, respectively. The RP-HPLC method has been successfully applied to estimate metronidazole and clonazepam in the well-known commercial pharmaceutical tablets and gave an excellent recovery> 98% for both drugs. The results of the method was compared with the standard pharmacopeial method for both drugs using statistical tests, which indicated no difference in accuracy between the methods

A New Simple Stability-Indicating RP-HPLC-PDA Method for Simultaneous Estimation of Triplicate Mixture of Sofosbuvir, Velpatasvir and Voxilaprevir in Tablet Dosage Form

A simple, rapid, selective, economical, accurate, reverse phase high performance liquid chromatography (RP-HPLC) was developed for simultaneous estimation of sofosbuvir, velpatasvir and voxilaprevir in its tablet dosage form. The separation was accomplished by means of a mobile phase of buffer and acetonitrile in the ratio of 50: 50 pumped at a flow rate of 1 ml/min along with 220 nm as a UV detection wavelength. The stationary phase used was column Waters C18 250 x 4.6 mm, 5m.μ. Sofosbuvir, velpatasvir and voxilaprevir were eluted at a retention times were 2.091 for sofosbuvir, 2.640 for velpatasvir and 3.349 min for voxilaprevir. Analytical method of the proposed RP-HPLC procedure was statistically validated with as per ICH guidelines by considering the parameters such as, system suitability, linearity ranges, precision, accuracy, specificity, and robustness, limit of detection and limit of quantification. The concentration ranges for linearity were 100–600, 25–150 and 25–150 ppm for sofosbuvir, velpatasvir and voxilaprevir respectively with correlation coefficients >0.999. The three drug samples were undergone degradation conditions of acidic and alkaline hydrolysis, oxidation, and photolysis, neutral and thermal degradation. The designed method proved to be stabilityindicating by resolution of the analytes from their forced-degradation products. The developed RP-HPLC method can be used for routine analysis of sofosbuvir, velpatasvir and voxilaprevir in combinational dosage form. The planned method finished by PDA as a tool for peak detection and purity confirmation.

Development and validation of an isocratic HPLC method for simultaneous determination of quaternary mixtures of antihypertensive drugs in pharmaceutical formulations

The objective of this study was to develop and validate an assay method for simultaneous determination of atenolol, furosemide, losartan, and spironolactone in pharmaceutical formulations. A reverse-phase high-performance liquid chromatography procedure was developed, using a Kinetex® C-18 column (100 mm × 4.6 mm, 2.6 μm). The mobile phase was composed of methanol—water (75:25 v/v, pH 3.0, adjusted with phosphoric acid), with a flow rate of 0.4 mL min−1. All drugs were separated in less than 5 min. The method was validated according to International Conference on Harmonization (ICH) and Association of Official Analytical Chemists (AOAC) guidelines. The method showed linearity in a concentration range of 0.75–12.0 μg mL−1 for atenolol (r = 0.9995), 0.30–12.00 μg mL−1 for furosemide (r = 0.9997), 0.45–12.00 μg mL−1 for losartan (r = 0.9995), and 0.45–12.0 μg mL−1 for spironolactone (r = 0.9999). The method also showed repeatability and precision. The three-day average intra-day precisions were 101.35 ± 0.74% for atenolol, 95.84 ± 1.44% for furosemide, 98.90 ± 1.16% for losartan, and 97.19 ± 0.18% for spironolactone. Similarly, the inter-day precisions were 101.34 ± 0.72% for atenolol, 95.84 ± 0.1.50% for furosemide, 98.90 ± 1.17% for losartan, and 97.19 ± 0.83% for spironolactone. The method accuracy was also tested and validated — in this case, the average recovery values were 100.18 ± 1.20% for atenolol, 99.83 ± 1.54% for furosemide, 100.07 ± 0.95% for losartan, and 99.94 ± 0.93% for spironolactone. Finally, the method was successfully applied in the simultaneous determination of atenolol, furosemide, losartan, and spironolactone in magisterial formulas, as well as in commercial pharmaceutical formulations.

Simultaneous Determination of Caffeine and Some Selected Polyphenols in Wuyi Rock Tea by High-Performance Liquid Chromatography

The primary taste and healthy benefits of tea are mainly attributed to tea polyphenols and caffeine. Due to very many kinds of flavonoid glycosides in tea and the lack of commercial standards of flavonoid glycosides, it is critical to develop a rapid and cheap method for determining flavonoid glycosides of tea. Contents of myricetin glycosides and quercetin glycosides in Wuyi Rock tea were determined by detecting contents of corresponding myricetin and quercetin. Optimizing hydrolysis conditions for hydrolyzing flavonoid glycosides to their corresponding flavonols including quercetin and myricetin in Wuyi Rock tea was a key technology for detecting contents of corresponding myricetin and quercetin. The results showed that hydrolysis at 2 mol/L HCl solution and at 90 °C for 1 h was an optimizing condition for hydrolyzing flavonoid glycosides to myricetin and quercetin in Wuyi Rock tea. Caffeine and seven kinds of polyphenols (GA, EGC, C, EGCG, EC, ECG, and CGA) in 20 samples of Wuyi Rock tea were simultaneously determined using a simple and fast reverse-phase high-performance liquid chromatography procedure coupled with photodiode array detector (RP-HPLC-PDAD). The results indicated that there were significant (P < 0.05) differences of ECG, CGA, ECG, and myricetin glycosides in 'Wuyi Rougui' and 'Wuyi Shuixian', which were credited with causing the difference in taste between these two cultivar of Wuyi Rock tea. The study may be useful for clarifying the cause of "cultivated varieties flavor" of Wuyi Rock tea.

Riluzole: Validation of Stability-Indicating HPLC, D1 and DD1 Spectrophotometric Assays

A stability-indicating reversed-phase high-performance liquid chromatographic assay procedure has been developed and validated for riluzole in the presence of alkaline and oxidative degradation products. The liquid chromatographic separation was achieved and compared isocratically on C18 Zorbax ODS and Poroshell 120 EC-C18 columns by using a mobile phase containing methanol-water, pH = 3.10 (70:30, v/v), at a flow rate of 1 mL/min and ultraviolet detection at 264 nm. The method was linear over the concentration ranges of 20-200 µg/mL (r = 0.9997) and 10-200 µg/mL (r = 0.9995). The limit of detection and quantitation for the two columns were 2 and 6 µg/mL and 1 and 3 µg/mL, respectively. Moreover, spectrophotometric methods were applied for the determination of riluzole in the presence of its oxidative degradation products by using first derivative spectrophotometry at 252.5 and 275.0 nm. The method was linear over the concentration range of 1-20 µg/mL (r = 0.9995 and 0.9996) at the studied wavelengths, with limits of detection and quantitation of 0.1 and 0.3 µg/mL. In addition, the first derivative ratio spectrophotometry (DD1) method was applied for the determination of riluzole in the presence of its alkaline degradation product at 252.0, 278.5 and 306.3 nm by using 100 µg/mL of alkaline degraded riluzole as a divisor; riluzole was additionally determined in the presence of its hydrogen peroxide oxidative degradation products at 252.5, 275.0 and 305.0 nm by using 200 µg/mL of oxidative degraded riluzole as a divisor. The DD1 method was linear over the concentration range of 1-20 µg/mL (r = 0.9996, 0.9995 and 0.9996 for the alkaline degradation product at the three studied wavelengths, respectively; and r = 0.9995, 0.9996 and 0.9995 for the oxidative degradation product at the three studied wavelengths, respectively), with limits of detection and quantitation of 0.1 and 0.3 µg/mL for both alkaline and oxidative degradation products. The two studied chromatographic and spectrophotometric methods were comparable and display the required accuracy, selectivity, sensitivity and precision to assay riluzole in bulk and pharmaceutical dosage forms. Degradation products resulting from the stress studies did not interfere with the detection of riluzole, which indicates that these are stability-indicating assays.

Comparative determination of miconazole, nystatin, hydrocortisone and neomycin by HPTLC/HPLC-DAD

Two new validated methods for the quantification of miconazole (MIC), nystatin (NYS), hydrocortisone acetate (HCA) and neomycin (NS) by high performance thin layer chromatography (HPTLC)-densitometry and reverse-phase high-performance liquid chromatography procedure coupled with photodiode array detector (RP-HPLC-DAD) were developed and compared. HPTLC separations were performed using a mobile phase, ethylacetate:methanol:glacial acetic acid (60:40:0.4, v : v : v ) for MIC, NYS, HCA and ethylacetate for NS. For HPLC-DAD analysis, using an isocratic elution system, separation of all compounds was achieved. Good resolution and quantization were achieved, accuracy and precision, as well as detection and quantitation limits of the two methods, were evaluated and compared. Excellent linearity was observed for all of the standard calibration curves, and the correlation coefficients were above 0.9997. HPTLC limits of quantitation were 15.37×10 -2 , 9.8×10 -2 , 13.32×10 -2 and 15.19×10 -2 µg/mL for MIC, NYS, HCA and NS respectively, whereas HPLC limits were 6.80×10 -2 , 6.56×10 -2 , 1×10 -1 and 10.16×10 -2 µg/band for MIC, NYS, HCA and NS respectively. In comparison with HPLC, HPTLC is less expensive and faster procedure, requiring 2-3 h to analyse 10-12 samples on a single plate. The developed methods were proved to be specific, robust and accurate for the determination of cited drugs in pharmaceutical preparations.

Simultaneous and accurate determination of water- and fat-soluble vitamins in multivitamin tablets by using an RP-HPLC method

In the present study, a reversed-phase high-performance liquid chromatographic (RP-HPLC) procedure was developed and validated for the simultaneous determination of seven water-soluble vitamins (thiamine, riboflavin, niacin, cyanocobalamin, ascorbic acid, folic acid, and p-aminobenzoic acid) and four fat-soluble vitamins (retinol acetate, cholecalciferol, α-tocopherol, and phytonadione) in multivitamin tablets. The linearity of the method was excellent (R² > 0.999) over the concentration range of 10 - 500 ng mL-1. The statistical evaluation of the method was carried out by performing the intra- and inter-day precision. The accuracy of the method was tested by measuring the average recovery; values ranged between 87.4% and 98.5% and were acceptable quantitative results that corresponded with the label claims.

A new HPLC method for the determination of tocopherol in rat plasma

Objective: The objective of the present work is the simplification of sample treatment for vitamin E measurements in rat plasma by high-performance liquid chromatography (HPLC), then the evaluation of the effect of cadmium treatment on the vitamin E level. Methods: A HPLC method to determine vitamin E was developed with direct extraction with n-hexane-dichloromethane. The dry residue was redissolved in methanol. The method employs a Supelco Discovery® C18 column and methanol/water (95:5, v/v) as the mobile phase. After being developed, the method was validated with a fluorescence detector. To examine the effects of exogenous cadmium on the plasma vitamin E concentrations, male Wistar rats (weighing 150 ± 20 g) were exposed for 48 h and 10 days to oral intake of 15 and 30 mg/L cadmium (as CdCl2) and vitamin E (10 mg/Kg of diet) simultaneously. Results: This method makes sample treatment easier, especially when working with a large number of samples. It has proved to be selective, linear, accurate and precise. Intoxication with cadmium was followed by a significant decrease in plasma vitamin E concentrations. The lowest level was obtained in the 30 mg/L dosing group after 10 days of cadmium administration. Conclusion: We describe a rapid reversed-phase (RP) HPLC procedure for the determination of vitamin E in plasma. This method might be useful in routine assessment because it saves on solvents and chromatographic time. Interestingly, α-tocopherol may play an important role in preventing oxidative stress induced by cadmium exposure by scavenging free radicals.

Rapid and simultaneous determination of antioxidant markers and caffeine in commercial teas and dietary supplements by HPLC-DAD

A simple and fast reverse-phase high-performance liquid chromatography procedure coupled with photodiode array detector (RP-HPLC-DAD) was developed and validated for the analysis of major catechins, proanthocyanidin (procyanidin B2) and caffeine in 25 different natural complex matrices containing Camellia sinensis L. and/or grape seed extracts, two popular plant extracts that have been widely used as natural antioxidants in various food and beverage applications. Using an isocratic elution system, separation of all compounds was achieved within 12min. Excellent linearity was observed for all of the standard calibration curves, and the correlation coefficients were above 0.9997. Limits of detection for all of the analyzed compounds ranged between 2.80×10−3 and 2.51×10−2μgmL−1; limits of quantitation ranged between 9.30×10−3 and 8.36×10−2μgmL−1. The developed method was found to be accurate and sensitive and is ideally suited for rapid, routine analysis of principal components in these well-known natural antioxidants.

RP-LC Determination of the cis-Isomer of Glimepiride in a Bulk Drug Substance

A rapid reversed-phase high-performance liquid chromatographic procedure is developed and validated for the resolution of the cis-isomer of 1-[[p-[2-(3-ethyl-4-methyl-2-oxo-3-pyrroline-1-carboxamido) ethyl] phenyl] sulfonyl]-3-(trans-4-methylcyclohexyl) urea, a dopamine agonist in bulk drugs. The cis-isomer and glimepiride were baseline resolved on a Waters Symmetry column (50 × 4.6 mm, 3.5 μm) using a mobile phase system containing water: tetrahydrofuran (75:25; v/v. The chromatographic resolutions between cis-isomer and glimepiride were found to be greater than two. The developed method was extensively validated and proved to be robust. The limit of detection and the limit of quantification of cis-isomer were 500 and 1,500 ng mL−1, respectively, for 10 μL injection volume. The percentage recovery of the cis-isomer ranged from 97.3 to 102.0 in bulk drug samples of glimepiride. Glimepiride sample solution and mobile phase were found to be stable for at least 48 h. The proposed method was found to be suitable and accurate for the quantitative determination of the cis-isomer in bulk drugs.

Development and validation of a liquid chromatography method for the simultaneous determination of α‐tocopherol, retinol and retinyl esters in human serum using a monolithic column for the monitoring of anticancer therapy side effects

Among other side effects, administration of anticancer agents is accompanied by manifestations of gastrointestinal toxicity and disturbances of antioxidant balance. The monitoring of these toxic effects in clinical practice is impeded by a dearth of reliable laboratory methods. Therefore, a simple and rapid reversed-phase high-performance liquid chromatography procedure for selective and sensitive determination of retinol, a-tocopherol, and retinyl esters (retinyl-palmitate and retinyl-stearate) in blood serum has been developed and presented in this study. A Series 200 LC HPLC instrument from Perkin Elmer (Norwalk, USA) with diode-array detector (DAD) was used for the analysis. Separations of retinol, alpha-tocopherol, retinyl-palmitate, and retinyl-stearate were performed using a Chromolith Performance RP-18e, 100 x 4.6 mm monolithic column from Merck (Darmstadt, Germany). Gradient elution was used at a flow rate of 3 mL/min; the mobile phase was methanol-water (95:5, v/v) for 0-2.1 min and methanol-2-propanol (60:40, v/v) for 2.1-4.9 min. The total time of analysis was 6 min. The injection volume was 20 microL and the analysis was performed at ambient temperature. Detection of retinol, alpha-tocopherol, and retinyl esters was carried out at 325, 295, and 330 nm, respectively. For practical assessment of the method, the vitamin A absorption test was performed on seven healthy controls as well as on six patients with non-small cell lung carcinoma or head and neck carcinoma previously treated by chemotherapy and/or radiotherapy, six patients with rectal carcinoma before chemoradiotherapy, four patients with gastrointestinal stromal tumor (GIST) before treatment with imatinib, and a breast cancer patient with chemotherapy-induced diarrhea. Present data demonstrate the feasibility of large scale HPLC determination of vitamin E, vitamin A, and retinyl esters in human serum using a silica monolithic column, and this method may represent a valuable aid in the laboratory monitoring of the toxicity of anticancer therapy.

PH/Organic Solvent Double-Gradient Reversed-Phase HPLC

A new reversed-phase high-performance liquid chromatographic (RP HPLC) procedure has been theoretically and experimentally established. The approach consists of the simultaneous development of a gradient of pH and of the organic modifier in the mobile phase. The proposed theoretical model of the pH/organic solvent double-gradient RP HPLC allows determination of both pK(a) and the lipophilicity parameter of the ionized and the nonionized form of the analyte and prediction of the retention times at specific separation conditions as well as bandwidth for all analytes. The model provides a rational basis for optimization of separation of ionizable analytes at any given chromatographic mode and analysis conditions. In addition, in the case of pH/organic solvent double-gradient RP HPLC, a compression of analyte peak and its reduced tailing can be expected.

High-Performance Liquid Chromatographic Analysis of Triterpenoids in Commercial Frankincense

A reversed-phase high-performance liquid chromatographic procedure has been developed: it is a simple and specific method for the determination of fifteen pentacyclic triterpenic compounds (a-boswellic acid, 3-O-acetyl-a-boswellic acid, b-boswellic acid, 3-O-acetyl-b-boswellic acid, a-amyrin, b-amyrin, lupeol, 3-epi-a-amyrin, 3-epi-b-amyrin, 3-epi-lupeol, a-amyrenone, b-amyrenone, lupenone, lupeolic acid and 3-O-acetyl-lupeolic acid) found in the most commonly traded frankincense, usually called ‘‘Eritrean-type’’ olibanum. In addition, the chromatographic comparison between fresh commercial resins and botanically certified ones was described in order to determine the geographical and/or the botanical origins of commercial frankincense. According to previous botanical studies, it appears difficult to make an unequivocal distinction between Boswellia carteri and B. sacra. On the other hand, Boswellia frereana (considered as a source of high-grade frankincense) shows a characteristic chromatogram and could be unambiguously distinguished from the other producing species of commercial frankincense. In a chemical point of view, Boswellia carteri and B. sacra were more especially characterized by the presence of lupeolic acid, boswellic acids and their respective O-acetyl derivatives, whereas 3-epi-lupeol was the major compound in B. frereana methanolic extracts.

Liquid chromatographic assay for riluzole in mouse plasma and central nervous system tissues

An isocratic, reversed-phase high-performance liquid chromatographic procedure (HPLC) was developed for determination of the neuroprotective agent riluzole in mice plasma, brain and spinal cord. The procedure is based on isolation of the compound and the internal standard from plasma and central nervous system tissues using a Bakerbond spe™ C8 cartridge, with satisfactory recovery and specificity. Separation was on a C18 column, coupled with an UV detector at 263 nm. The assay was linear over a wide range, with a lower limit of quantification of 100 ng ml −1 or g −1 using 0.1 ml of plasma and about 100 mg of brain tissue. The precision and accuracy were within the acceptable limits for an HPLC assay. The method is currently used to support pharmacological studies of the activity of riluzole when given in combination with other potential neuroprotective agents in an animal model of familiar amyotrophic lateral sclerosis (SOD1-G93A transgenic mice).

Analysis of Vegetable Oils by High-Performance Liquid Chromatography

A reversed-phase high-performance liquid chromatographic procedure is proposed to monitor the fatty-acid and triglyceride composition of sunflower-seed oil. The procedure uses a column packed with Diasfer-110-C18 (6 μm), a 2 : 8 mixture of acetonitrile and acetone as an eluent, and refractometric detection. Analyses that use simple normalization and normalization with correction factors that take into account the difference between calculated refraction coefficients of different triglycerides are compared. It is shown that the proposed procedure can be used for the quantitative determination of total triglycerides in various vegetable oils as well.

A rapid high-performance liquid chromatographic method for the simultaneous quantitation of aspirin, salicylic acid, and caffeine in effervescent tablets.

A rapid reversed-phase high-performance liquid chromatographic procedure is developed and validated for the simultaneous quantitation of aspirin, salicylic acid, and caffeine extracted from an effervescent tablet. The method uses a Hypersil C18 column (5 micro m, 15 cm x 4.6 mm) for an isocratic elution in a water-methanol-acetic acid mobile phase at a wavelength of 275 nm. The tablets' buffering effects and acid neutralizing capacity require an extraction solvent of methanol-formic acid. The range of linearity for aspirin is 0.5-1.25 mg/mL, caffeine 0.065-0.195 mg/mL, and salicylic acid 0.4-6.0% of aspirin. The overall recovery is 100.2%, 100.7%, and 99.2% for aspirin, caffeine, and salicylic acid, respectively. Under the conditions of the method, aspirin, caffeine, and salicylic acid are adequately resolved with proper peak symmetry in less than 7 min.

Liquid chromatographic determination of minocycline in brain-to-plasma distribution studies in the rat

An isocratic reversed-phase high-performance liquid chromatographic procedure was developed for the determination of minocycline in rat plasma and brain and applied to brain-to-blood (plasma) distribution studies. The procedure is based on isolation of the compound and the internal standard (either demeclocycline or tetracycline may be used) from plasma and brain constituents using the Oasis HLB cartridge, with satisfactory recovery and specificity, and separation on a Symmetry Shield RP8 (15 cm×4.6 mm, 3.5 μm) column coupled with a UV detector set at 350 nm. The assay was linear over a wide range, with a lower limit of quantification of 50 ng ml −1 or g −1, using 0.2 ml of plasma and about 200 mg of brain tissue. Precision and accuracy were acceptable. In the rat minocycline crossed the blood–brain barrier slowly, achieving mean brain concentrations between 30 and 40% of the equivalent systemic exposure, regardless of the dose and route of administration.

Reversed-phase high-performance liquid chromatography procedure for the simultaneous determination of S-adenosyl- l-methionine and S-adenosyl- l-homocysteine in mouse liver and the effect of methionine on their concentrations

An improved reversed-phase high-performance liquid chromatography (HPLC) procedure with ultraviolet detection is described for the simultaneous determination of S-adenosyl- l-methionine (SAM) and S-adenosyl- l-homocysteine (SAH) in mouse tissue. The method provides rapid resolution of both compounds in a 25-μl perchloric acid extract of the tissue. The limits of detection in 25-μl injection volumes were 22 and 20 pmol for SAM and SAH, respectively. The limits of quantitation in 25-μl injection volumes were 55 and 50 pmol for SAM and SAH, respectively, with recovery consistently >98%. The assay was validated over linear ranges of 55–11 000 pmol for SAM and 50–10 000 pmol for SAH. The intra-day precision and accuracy were ≤6.4% relative standard deviation (RSD) and 99.9–100.0% for SAH and ≤6.7% RSD and 100.0–100.1% for SAM. The inter-day precision and accuracy were ≤5.9% RSD and 99.9–100.6% for SAH and ≤7.0% RSD and 99.5–100.1% for SAM. Compared to earlier procedures, the HPLC method demonstrated significantly better separation, detection limit and linear range for SAM and SAH determination. The assay demonstrated applicability to monitoring in mice the time-course of the effect of methionine on SAM and SAH levels in the liver. Administering methionine to mice increased by 10-fold the liver concentration of SAM and SAH within 2 h, which then rapidly decreased to the control levels by 8 h. This indicated that methionine was promptly converted to SAM and then rapidly catabolized into SAH. Thus, the metabolism of methionine to SAM should be considered in the supplementation of methionine to maintain SAM levels in the body.

Determination of the molecular weight and other physicochemical properties of poly(bubylene glycol) 1000 by gradient RPHPLC and measurement of the ELSD, UV, and fluorescence responses

Molecular weight and other physicochemical data for poly(butylene glycol) 1000 (PBG 1000) have been determined by use of a new, highly efficient, gradient reversed-phase high-performance liquid-chromatographic (RPHPLC) procedure. Separation of the native material or its α, ω-bis(1-naphthylurethane) derivative was achieved on a C18 stationary phase with a ternary acetonitrile-water-tetrahydrofuran mobile phase. Detection was achieved by measurement of the signal response from evaporative light scattering (ELSD), UV, and fluorescence (FD) detection. Proof that all the oligomers contained in the sample had been separated by the method was obtained by liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry (LC-ESI-TOFMS). It was also confirmed by this technique that di(butylene glycol) is the lowest homologue in the sample. Although the dimer was also observable in the HPLC-UV trace of the PBG 1000 α, ω-bis(1-naphthylurethane) derivative, it was obviously too valatile to be seen in the ELSD trace; tri(butylene glycol) was, nevertheless, still recognizable with sufficient signal intensity. Because all the homologues were separated to baseline, the method was used to calculate the number- and weight-average molecular weights,M n andM w, both from peak areas and from peak heights. The best fit to data obtained from end-group titration were obtained from calculations based on the HPLC-UV response;M n values of 948 and 937 were obtained from peak heights and areas, respectively.M n andM w values calculated from the ELSD trace obtained from native PBG 1000 were substantially (ca 10%) lower that those calculated from the UV trace obtained from the α, ω-bis(1-naphthylurethane) derivative. Similar differences were also discovered by comparing theM n andM w values obtained from UV and FD-values were approximately 20% higher for FD. When the retention times of individual oligomers, from either ELSD of the native sample or from UV-FD of the α,ω-bis(1-naphthylurethane) derivative, were plotted against the number of repeat units,P, the ELSD curve approaches the UV-FD curve at values ofP of approximately 60. This observation can be explained by the pronounced contribution of the hydrophobic end-groups to the overall retention of PBG 1000; the effect of this decreases with increasing chain length and becomes nearly independent ofP only for a very high degree of polymerization.