Proposed High-performance Liquid Chromatography Method Research Articles

Quantitative determination of caffeine in different matrices

Caffeine is odorless, bitter taste substance which can be naturally found in coffee, cocoa, tea leaves, and is intentionally added in food and pharmaceutical products. It can also be found in surface water in small concentrations where is often used as an excellent indicator of human waste. The aim of the work is determination of caffeine content in food, beverages, analgesics and surface water using solidphase extraction followed by high-performance liquid chromatography (HPLC). Caffeine content was determined in 12 commercial tea and coffee products, non-alcoholic energy drinks and food, 5 combined preparations of analgesics and the Danube samples collected from 7 representative locations. The results showed that caffeine content in food ranged 5,6-158 mg/100 g, tea samples 24,71-30,81 mg/100 ml, coffee samples 1328-3594 mg/100 g, energy drinks 9,69-30,79 mg/100 ml and in the Danube samples 15,91-306,12 ng/l. Caffeine content in combined commercial formulations of non-narcotic analgesics of all brands did meet specifications. The data suggested that the proposed HPLC method can be used for routine determination and control of caffeine content in different matrices.

Validation of high performance liquid chromatography method for determination of meloxicam loaded PEGylated nanocapsules

abstract A method to ensure that an analytical method will produce reliable and interpretable information about the sample must first be validated, making sure that the results can be trusted and traced. In this study, we propose to validate an analytical high performance liquid chromatography (HPLC) method for the quantitation of meloxicam loaded PEGylated nanocapsules(M-PEGNC). We performed a validation study, evaluated parameters including specificity, linearity, quantification limit, detection limit, accuracy, precision and robustness. PEGylated nanocapsules were prepared by interfacial deposition of preformed polymer, and the particle size, polydispersity index, zeta potential, pH value and encapsulation efficiency were characterized. The proposed HPLC method provides selective, linear results in the range of 1.0-40.0 μg/mL; quantification and detection limits were 1.78 μg/mL and 0.59 μg/mL, respectively; relative standard deviation for repeatability was 1.35% and intermediate precision was 0.41% and 0.61% for analyst 1 and analyst 2, respectively; accuracy between 99.23 and 101.79%; robustness between 97.13 and 98.45% for the quantification of M-PEGNC. Mean particle diameters were 261 ± 13 nm and 249 ± 20 nm, polydispersity index was 0.15 ± 0.07 and 0.17 ± 0.06, pH values were 5.0 ± 0.2 and 5.2 ± 0.1, and zeta-potential values were -37.9 ± 3.2 mV e -31.8 ± 2.8 mV for M-PEGNC and placebo(B-PEGNC), respectively. In conclusion, the proposed analytical method is suitable for the quality control of M-PEGNC. Moreover, suspensions showed monomodal size distributions and low polydispersity index indicating high homogeneity of formulations with narrow size distributions, and appropriate pH and zeta potential. The extraction process was efficient for release of meloxicam from nanostructured systems.

Stability of aspartame and neotame in pasteurized and in-bottle sterilized flavoured milk

Analytical high performance liquid chromatography (HPLC) conditions were standardized along with the isolation procedure for separation of aspartame and neotame in flavoured milk (pasteurized and in-bottle sterilized flavoured milk). The recovery of the method was approximately 98% for both aspartame and neotame. The proposed HPLC method can be successfully used for the routine determination of aspartame and neotame in flavoured milk. Pasteurization (90°C/20min) resulted in approximately 40% loss of aspartame and only 8% of neotame was degraded. On storage (4–7°C/7days) aspartame and neotame content decreased significantly (P<0.05) from 59.70% to 44.61% and 91.78% to 87.18%, respectively. Sterilization (121°C/15min) resulted in complete degradation of aspartame; however, 50.50% of neotame remained intact. During storage (30°C/60days) neotame content decreased significantly (P<0.05) from 50.36% to 8.67%. Results indicated that neotame exhibited better stability than aspartame in both pasteurized and in-bottle sterilized flavoured milk.

Detection of honey adulteration with starch syrup by high performance liquid chromatography

According to saccharide profile comparison between starch syrups and pure honeys analysed through high performance liquid chromatography (HPLC), a characteristic peak was found at 15.25min retention time in HPLC chromatogram of syrup, but no peak was observed at the same retention time in chromatogram of pure honeys. This characteristic peak for syrup was identified as an overlapping peak of oligosaccharides with more than 5 degree of polymerisation (DP) based on HPLC chromatogram comparison between starch syrup and a series of standard mono-, di- and oligosaccharides of 3–7 DP. Additionally syrup content correlated linearly with the height of the characteristic peak of syrup under different slope in two ranges 2.5–7.5% and 10–100%, respectively. Therefore, the characteristic peak at 15.25min retention time can serve as a syrup indicator in HPLC analysis of the adulterated honeys. This new HPLC method for honey adulteration detection was further applied in an authenticity inspection on more than 100 commercial honeys. In addition to the improved accuracy of honey adulteration detection, the proposed HPLC method was simple, low cost and easy practice for honey product quality control by government department considering the popularity of HPLC device and technology.

Simultaneous quantification of furanocoumarins from Aegle marmelos fruit pulp extract.

Aegle marmelos Correa (Rutaceae) is a prominent species in the Sub-Himalayan region, also found in central and south India, and has been widely used as remedy for diarrhea and dysentery. Unripe fruits show antiviral activity and prove to be a potent hypoglycemic agent. In this study, a high-performance liquid chromatography (HPLC) method for the simultaneous quantification of major furocoumarin components marmelosin, psoralen and bergapten in the extract from fruit pulp of A. marmelos has been developed. Components were found in the range of 5-6.5, >0.4-0.75 and >0.25 mg/g of dried fruit weight, respectively. The method was validated for linearity, precision, accuracy, robustness, limit of detection and limit of quantification. Linearity was determined over the range of 5-40 µg/mL (r > 0.965). Intra- and interday precision showed a relative standard deviation of <2.5%. The accuracy of the method was determined by a spike recovery study, and the average recoveries were 99.46, 101.04 and 100.8% for marmelosin, psoralen and bergapten, respectively. The proposed HPLC method was found to be simple, precise and specific and can be used simultaneously for the routine quality control of raw materials of A. marmelos fruit extracts and their products, and also other products containing these markers.

Isolation and HPLC method development of azafrin from Alectra parasitica var. chitrakutensis

This study was undertaken to isolate and quantify azafrin in Alectra parasitica (Scrophulariaceae) rhizomes. A simple method for the isolation of carotenoid, azafrin, involves solvent extraction of the dried rhizome powder using a single solvent and further purification by recrystallisation. The structure of the compound was elucidated and confirmed by thin-layer chromatography, infrared spectroscopy, mass spectroscopy and nuclear magnetic resonance spectral analysis. A specific and rapid reversed-phase high-performance liquid chromatography (HPLC) method was developed for the analysis of azafrin. The method was validated for accuracy, precision, linearity and specificity. Validation revealed that the method is specific, accurate, precise, reliable and reproducible. The proposed HPLC method can be used for the identification and quantitative analysis of azafrin in A. parasitica rhizomes.

Rapid preparative extraction and determination of major organic acids in honeysuckle (Lonicera japonica Thunb.) tea

In this study, ionic liquid based microwave-assisted extraction (IL-MAE) followed by high-performance liquid chromatography (HPLC) was developed for determination of major organic acids in honeysuckle tea. The results suggest that varying the anion and cation had significant effects on the preparative extraction efficiency; aqueous solution of [C6mim]Br was selected as solvent. The optimized extraction parameters were obtained as follows: IL concentration 0.5M, temperature 50°C, solvent to material ratio 20:1mL/g and duration 9min. Moreover, principal component analysis (PCA) was used to analyze the significances of these factors for IL-MAE and their interactions, and IL concentration was proved to be the most significant parameter. Compared with conventional sample preparation method, the content of organic acids in analytical sample was enhanced about 1.5-fold using IL-MAE in a shorter duration. In addition, a kinetic model of the preparative extraction process was established to fit the experimental data (R2>0.99). The proposed HPLC method was successfully applied for determination of 5 organic acids in honeysuckle tea. The present research supported necessary data for sample preparation and quality analysis of honeysuckle tea.

Analysis of four anticoagulants rodenticides in foodstuff and body fluids by high-performance liquid chromatography with photodiode-array detector

A sensitive high-performance liquid chromatography (HPLC) method was developed for the determination of four anticoagulant rodenticide residues (warfarin, coumatetralyl, crimidine, and bromadiolone) in foodstuffs and body fluids. The conditions of accelerated solvent extraction (ASE) and HPLC were optimized. Good linearity was achieved with a correlation coefficient (r) of 0.9999. The limits of quantification for the four rodenticides ranged from 2.2 μg kg−1 (crimidine) to 13 μg kg−1 (bromadiolone) for solid samples and from 0.02 μg mL−1 (crimidine) to 0.15 μg mL−1 (bromadiolone) for liquid samples. The intra- and inter-day relative standard deviations (RSDs) of the four analytes at two spiked levels were in the range of 0.3–1.1% and 0.5–1.6%, respectively. The mean recoveries for warfarin, coumatetralyl, crimidine, and bromadiolone at the two spiked levels were in the range of 94–106% with RSDs of 0.3–1.1% for the rice and corn samples, and 87–108% with RSDs of 0.3–0.8% for the whole blood and urine samples. The proposed method has characteristics of high precision, sensitivity and accuracy as well as low consumption of the reagents. The proposed HPLC method was successfully applied for the analysis of the four anticoagulant rodenticides in rice, corn, whole blood, and urine samples.

DETERMINATION OF LAMOTRIGINE IN TABLETS USING HPTLC, HPLC, AND DERIVATIVE SPECTROPHOTOMETRY METHODS

The determination of lamotrigine in tablets using high-performance thin-layer chromatography (HPTLC), high-performance liquid chromatography (HPLC), as well as direct, first, second, and third derivative ultraviolet spectrophotometry methods are described. HPTLC method was performed using silica plates, a mobile phase composed of toluene:acetone:ammonia (3:7:0.5, v/v/v), and a densitometric detection at 312 nm. Moreover, quantification was achieved in the concentration range of 0.25–10.0 µg/spot and with good precision (RSD = 2.19%) and recovery (97.92%), using a nonlinear calibration curve by fitting to y = a + blnx. HPLC method was performed using a Luna C18 column and isocratic elution mode with a mobile phase composed of methanol: phosphate buffer (25:75, v/v) and delivered at a flow rate 1.2 mL/min. Proposed HPLC method provided good results of precision (RSD = 1.86%) and recovery (101.51%) in the concentration range of 0.25–7.5 µg/mL using linear y = a + bx regression analysis. For the UV-derivative spectrophotometry method, all derivatives and wavelengths studied proved accurate linearity, precision (RSD = 0.96–1.87%), and recovery (96.74–101.90%). Moreover, no interference was found from tablet excipients at the selected wavelength and assay procedures. The developed methods were found to be validated and showed good precision and reproducibility.

Validated RP-HPLC method for the simultaneous determination of amlodipine besylate, valsartan, and hydrochlorothiazide in bulk and in pharmaceutical formulation

Summary A reverse-phase high-performance liquid chromatographic (RP-HPLC) method was developed for the simultaneous estimation of amlodipine besylate (AMB), valsartan (VAL), and hydrochlorothiazide (HCT) in pharmaceutical formulation using RP-C18 column. The mobile phase (acetonitrile:methanol:50 mM phosphate buffer adjusted to pH 3 with orthophosphoric acid) was pumped at a flow rate of 1.0 mL min−1 in the ratio of 20:50:30% v/v and the eluents were monitored at 239 nm. Linearity was obtained in the concentration range of 0.5–5 μg mL−1 for AMB, 4–40 μg mL−1 for VAL, and 1–10 μg mL−1 for HCT. The method was validated as per International Conference on Harmonization (ICH) guidelines and statistically. The method was validated for accuracy and precision. For precision, the coefficient of variance (COV) was found to be 0.3794, 0.1703 and 0.0578, and for accuracy, it was found to be 0.6351, 0.7688 and 1.1305 for AMB, VAL, and HCT, respectively. The COV values for all the drugs were found to be less than 2%, indicating high degree of precision and accuracy of the proposed high-performance liquid chromatographic (HPLC) method. Owing to its simplicity, rapidness, high precision and accuracy, the proposed HPLC method can be applied for determining AMB, VAL, and HCT in bulk and in pharmaceutical dosage form.

A validated UV-HPLC method for determination of chlorogenic acid in Lepidogrammitis drymoglossoides (Baker) Ching, Polypodiaceae

Background:Lepidogrammitis drymoglossoides (Baker) Ching (L. drymoglossoides), a member of the Polypodiaceae family, was used in the treatment of numerous diseases. However, none of the potential ingredients and the quality control methods concerning this plant medicine was pronounced.Objective:To identify chlorogenic acid (CGA) from L. drymoglossoides and develop a high performance liquid chromatography (HPLC) assay of CGA.Materials and Methods:UV, TLC, and HPLC were utilized to identify the phytochemicals of L. drymoglossoides and determine the CGA content, respectively. The HPLC conditions were as following: a Phenomenex Luna C18 (2) (250 × 4.6 mm i.d.; 5 μm particle size; 100 Å pore size) column; the mobile phase of the mixture of acetonitrile and 0.5% aqueous phosphoric acid (11.5:88.5 v/v); the flow rate of 1.0 mL/min and determination wavelength of 327 nm.Results:The proposed HPLC method has been developed and validated. The calibration curve was y = 28328x + 16610 (R2 = 0.9997). The intra-day and inter-day precision and intermediate precision were validated with the RSD less than 5%. The mean recovery rate of the method ranged from 95% to 104%, with the RSD less than 5%. The LOD and LQD values were 0.049 and 0.132 mg/L, respectively. The content of CGA in L. drymoglossoides approximately reached 0.24% (v/v) by the proposed extraction and determination methods.Conclusion:The assay method was simple, convenient, and accurate to the quantification of CGA and can be used for the quality control of the herb.

High performance liquid chromatographic method development for simultaneous analysis of doxofylline and montelukast sodium in a combined form

Aim: Some literatures revealed that the high performance liquid chromatography (HPLC) method for single component or multicomponent analysis of montelukast sodium with other drugs. However, these methods deals with time consuming, so it is necessary to develop a cost-effective and less time consuming method for the estimation of doxofylline and montelukast sodium in combined pharmaceutical formulation. Materials and Methods: The separation was performed on an inertsil C8 (5 μm, 4.6 × 250 mm) column in isocratic mode with the mobile phase consisting a mixture of methanol and sodium phosphate buffer (75:25 v/v, pH 6.5 adjusted with orthophosphoric acid). The mobile phase was pumped at a flow rate of 1 mL min-1 and eluents were monitored at 230 nm. Results: The selected chromatographic conditions were found to separate doxofylline (retention time = 3.4 min) and montelukast sodium (retention time = 5.5 min) with a resolution of 5.47. The proposed HPLC method was validated with respect to linearity, accuracy, repeatability, specificity, robustness, and ruggedness as per International Conference on Harmonisation guidelines Q2(R1), November 2005 (Validation of Analytical Procedures: Text and Methodology). The percentage recoveries for doxofylline and montelukast sodium ranged from 98.1% to 101.7% and 98.2 to 101.9%, respectively, which indicated that the above method was enough accurate and precise. Conclusions: Hence, it was concluded that the developed method is suitable for routine analysis of these combination due to its less analysis time.

Determination of N‐acetylaspartic acid concentration in the mouse brain using HPLC with fluorescence detection

The concentration of brain N-acetylaspartic acid (NAA) in mice was determined by high-performance liquid chromatography (HPLC) using fluorescence detection after pre-column derivatization with 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole (DBD-ED). Six different brain parts, namely, the prefrontal cortex, olfactory bulb, nucleus accumbens, striatum, cerebellum and hippocampus, of male C57BL6/J mice, were investigated. The NAA concentration (nmol/mg protein) was highest in the olfactory bulb (58.2 ± 4.0, n = 8) and lowest in the hippocampus (42.8 ± 1.6, n = 8). The proposed HPLC method with fluorescence detection was successfully used to determine the NAA concentration in each investigated brain area.

Selective determination of ubiquinone in human plasma by HPLC with chemiluminescence reaction based on the redox cycle of quinone

Ubiquinone is an important biologically active compound in the living body. The determination of ubiquinone in human plasma is useful for the investigation of bioavailability of ubiquinone and for early diagnosis of several diseases. Therefore, we developed a high-performance liquid chromatography (HPLC) with chemiluminescence detection method for the analysis of ubiquinone in plasma samples. The method is based on luminol chemiluminescence detection of super oxide anion that is generated by the redox cycle reaction between ubiquinone and dithiothreitol. The HPLC system involved an octyl column with a mobile phase of methanol. Ubiquinone eluted from the column was mixed with dithiothreitol and luminol solutions simultaneously, and generated chemiluminescence was monitored by chemiluminescence detector. The calibration curve for standard ubiquinone solution was linear from 0.09 to 43.2μg/mL (0.45-216ng on column) with the correlation coefficient of 0.999, and the detection limit (S/N = 3) was 26ng/mL (130pg on column). Using the proposed HPLC method, the peak of ubiquinone in human plasma could be clearly detected on the chromatogram without any interference from plasma components.

Development and validation of an HPLC method for simultaneous determination of trimethoprim and sulfamethoxazole in human plasma

The combination of trimethoprim (TMP) and sulfamethoxazole (SMX) is used in the treatment of many common infections such as urinary, respiratory and gastrointestinal tract infections. The aim of this study was to determine TMP and SMX simultaneously in human plasma samples by high performance liquid chromatography (HPLC) using antipyrine as the internal standard. Separation of the compounds was achieved on a reverse-phase C8 column packed with 5 microm dimethyl octadecylsilyl bonded amorphous silica (4.6 mm x 250 mm) column using a mobile phase consisted of potassium hydrogen phosphate, acetonitrile, methanol and water adjusted to pH 6.2. The mobile phase was delivered at a flow rate of 1 mL min- and the effluent was monitored using Max plot technique at 25 derees C. Retention times were 5 min for TMP, 7 min for antipyrine and 9 min for SMX. Quantitation limits were 10 ng mL(-1) for TMP and 50 ng mL(-1) for SMX. Our findings indicated that the developed HPLC method was precise, accurate, specific and sensitive for simultaneous determination of TMP and SMX. Proposed HPLC method was successfully applied for the analysis of TMP and SMX in human plasma after oral administration of a co-trimoxazole tablet to human volunteers.

Application of high performance liquid chromatography to the determination and validation of levodopa in methanolic extract of Mucuna utilis

A sensitive, precise and accurate high-performance liquid chromatographic (HPLC) method has been developed for the analysis of levodopa in methanolic extract of Mucuna utilis. The method utilises sample preparation step followed by separation on a Eurosphere C18, 250 × 4.0 mm, 5 μm particle size column, using methanol and 0.5% v/v acetic acid in the ratio of 70:30 v/v as the mobile phase. Analysis of levodopa was carried out in the absorbance mode at 284 nm. The method was validated in terms of linearity, precision (inter and intra day), accuracy, Limit of Detection (LOD) and Limit of Quantitation (LOQ). The proposed HPLC method was found to be precise, specific, accurate and can be used for the identification and quantitative determination of levodopa in herbal extracts. Key words: High-performance liquid chromatography, levodopa, method development and validation, Mucuna utilis

Direct high-performance liquid chromatography enantioseparation of terazosin on an immobilised polysaccharide-based chiral stationary phase under polar organic and reversed-phase conditions

High-performance liquid chromatography (HPLC) enantioseparation of terazosin (TER) was accomplished on the immobilised-type Chiralpak IC chiral stationary phase (CSP) under both polar organic and reversed-phase modes. A simple analytical method was validated using a mixture of methanol–water–DEA 95:5:0.1 (v/v/v) as a mobile phase. Under reversed-phase conditions good linearities were obtained over the concentration range 8.76–26.28 μg mL −1 for both enantiomers. The limits of detection and quantification were 10 and 30 ng mL −1, respectively. The intra- and inter-day assay precision was less than 1.66% (RSD%). The optimised conditions also allowed to resolve chiral and achiral impurities from the enantiomers of TER. The proposed HPLC method supports pharmacological studies on the biological effects of the both forms of TER and analytical investigations of potential drug formulations based on a single enantiomer. At the semipreparative scale, 5.3 mg of racemic sample were resolved with elution times less than 12 min using a mobile phase consisting of methanol–DEA 100:0.1 (v/v) and both enantiomers were isolated with a purity of ≥99% enantiomeric excess (ee). The absolute configuration of TER enantiomers was assigned by comparison of the measured specific rotations with those reported in the literature.

Development Of a Simple HPLC Method for the Quantitation of Artemisinin in Artemisia Annua Herb

A high-performance liquid chromatographic (HPLC) method has been developed for the determination of Artemisinin in Artemisia annua Herb. The HPLC separation was carried out in isocratic mode using C18, sunfire column (4.6 × 250 mm, 5 μm particle size) with a mobile phase composed of acetonitrile : water (85:15 v/v) at a flow rate of 1.0 ml/min. The detection was monitored at 216 nm. The method validation parameters showed good results for linearity, precision, accuracy, specificity, and in recovery studies. The calibration curve for Artemisinin was found linear from the range of 500 to 1200 μg/ml. The interday and intraday studies (relative standard deviation) was obtained within the limits. The proposed HPLC method is precise, accurate and rapid for determination of Artemisinin in Artemisia annua Herb.

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 type3 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 × 10 2 ± 9.2 nmol/mg protein, respectively ( n = 6), while those in the hippocampus were 6.8 ± 0.48 and 1.9 × 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).

Separation and quantification of component monosaccharides of the tea polysaccharides from Gynostemma pentaphyllum by HPLC with indirect UV detection

A reversed-phase high-performance liquid chromatographic (HPLC) method is described for the simultaneous determination of aldoses and uronic acids. The separation was carried out on a RP-C 18 column (4.6 mm i.d. × 250 mm, 5 μm, Venusil, USA) using precolumn derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) and UV detection at 250 nm, and the 10 PMP derivatives of mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, xylose, galactose, arabinose and fucose were baseline separated within 40 min. Furthermore, the described method was applied to the quantitative analysis of component monosaccharides in the water-soluble polysaccharides extracted from Gynostemma pentaphyllum Makino tea and the result showed that the tea polysaccharide was a typical heteropolysaccharide and consisted of mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, xylose, galactose and arabinose in the molar contents of 16.3, 10.3, 47.1, 5.6, 24.0, 128.4, 25.0, 101.4 and 71.1 μM, respectively. Quantitative recoveries of the component monosaccharides in the tea polysaccharide were in the range of 94.6–108.0% and the RSD values were lower than 4.9%. The results demonstrated that the proposed HPLC method was precise and practical for the analysis of the G. pentaphyllum tea polysaccharide.