Ion-pair Reversed-phase High-performance Liquid Research Articles

Validation of an Ion-Pair Reverse Phase High-Performance Liquid Chromatography Method for the Detection of Major Components and Related Substances in Diquafosol Sodium Eye Drops.

A simple, feasible, isocratic elution, and stable reversed-phase high performance liquid chromatography method was established and verified. The chromatographic conditions are as follows: EF-C18H, 4.6 × 250 mm, 5 μm column; column temperature 30 °C; for the mobile phase 27.2 g of KH2PO4 and 8.5 g of tetrabutylammonium hydrogen sulfate were taken, 2500 mL of water was added to dissolve, and the pH was adjusted to 6.7 with phosphoric acid:methanol solution with a ratio of 84:16 (V:V). The flow rate was 1.0 mL/min; the injection volume was 10 μL; and the wavelength was 262 nm. According to the current ICH guidelines, the developed method was verified, and the system suitability, specificity, LOD, LOQ, linearity, range, accuracy, repeatability, durability, and solution stability of the proposed method were verified. The validation results demonstrated that the LOQ for the method was 0.05% and the LOD was 0.02%. The content was detected within the concentration range of 300 to 900 μg/mL. The relationship between concentration and measurement was linear, with an r2 of >0.999. The concentration of impurities ranged from 0.3 to 4.5 μg/mL. A good linear correlation was observed within the range of g/mL, with a coefficient of determination r2 greater than 0.999. The accuracy and repeatability met the specified criteria.

Simultaneous quantification of multiple RNA cargos co-loaded into nanoparticle-based delivery systems

Robust, sensitive, and versatile analytical methods are essential for quantification of RNA drug cargos loaded into nanoparticle-based delivery systems. However, simultaneous quantification of multiple RNA cargos co-loaded into nanoparticles remains a challenge. Here, we developed and validated the use of ion-pair reversed-phase high-performance liquid chromatography combined with UV detection (IP-RP-HPLC-UV) for simultaneous quantification of single- and double-stranded RNA cargos. Complete extraction of RNA cargo from the nanoparticle carrier was achieved using a phenol:chloroform:isoamyl alcohol mixture. Separations were performed using either a C18 or a PLRP-S column, eluted with 0.1 M triethylammonium acetate (TEAA) solution as ion-pairing reagent (eluent A), and 0.1 M TEAA containing 25 % (v/v) CH3CN as eluent B. These methods were applied to quantify mRNA and polyinosinic:polycytidylic acid co-loaded into lipid-polymer hybrid nanoparticles, and single-stranded oligodeoxynucleotide donors and Alt-R CRISPR single guide RNAs co-loaded into lipid nanoparticles. The developed methods were sensitive (limit of RNA quantification < 60 ng), linear (R2 > 0.997), and accurate (≈ 100 % recovery of RNA spiked in nanoparticles). Hence, the present study may facilitate convenient quantification of multiple RNA cargos co-loaded into nanoparticle-based delivery systems.

Advancements in the characterisation of oligonucleotides by high performance liquid chromatography-mass spectrometry in 2021: A short review.

The first oligonucleotide therapeutic was approved by the Food and Drug Administration in 1998, and since then, 12 nucleic acids have been commercialised as medicines. To be approved, the oligonucleotides need to be identified and characterised as well as its related impurities. Different methods exist, but the most commonly used is ion-pairing reversed-phase liquid chromatography with tandem mass spectrometry. The separation obtained depends on the mobile phase and column used. Other methods have been developed, notably by using hydrophilic interaction chromatography and two-dimensional high performance liquid chromatography. Furthermore, ion-pairing reversed-phase high performance liquid chromatography ultra-violet spectroscopy detection and mass spectrometry has been optimised for the analysis of methylated nucleobases due to the utilisation of this modification in the drugs. This review covers the recent advancements in the analysis and characterisation of oligonucleotides in 2021 by high performance liquid chromatography mass spectrometry, notably by hydrophilic interaction chromatography and two-dimensional liquid chromatography but also the different parameters that influence the analysis by ion-pairing reversed-phase high performance liquid chromatography, the characterisation of methylated nucleobases, and the recent software developed for oligonucleotides.

Innovated formulation of oseltamivir powder for suspension with stability study after reconstitution using a developed ion-pair reversed phase high-performance liquid chromatography method.

ABSTRACTOseltamivir is an antiviral neuraminidase inhibitor used for the treatment and prophylaxis of influenza infection with viruses A and B. The mechanism of oseltamivir antiviral activity is by inhibiting the activity of the viral neuraminidase enzyme present on the surface of the virus, which stops viral replication and infectivity. Oral suspensions of oseltamivir phosphate are dispensed orally capsules and suspension. However, the use of oral suspension for pediatric administration is preferable and is prepared as a powder for suspension. The reconstituted suspension degrades rapidly within a few days. The objective of this work is to establish a stable formulation of oseltamivir phosphate as a suspension and to assure the stability conditions for prolonged use after reconstitution in aqueous form. In addition, this required formulation should maintain a high rate of dissolution, which subsequently leads to higher bioavailability. In this study, oseltamivir forms an inclusion complex with the natural and safe polymer hydroxypropyl beta-cyclodextrin, which resembles a host because its structural cavity carries the oseltamivir molecule in the aqueous preparation and provides a protective property against environmental challengers. In addition, a high-performance liquid chromatography (HPLC) stability-indicating method of analysis has been developed using an ion-pair reversed-phase HPLC technique that is validated for precision, accuracy, reproducibility, and specificity for the determination of oseltamivir in suspension. The results of this work show the relatively long shelf life of the reconstituted oseltamivir oral powder for suspension in the new pediatric formulation, and the developed HPLC method was precisely suitable for stability study.

The glutamyl tail length of the cofactor F420 in the methanogenic Archaea Methanosarcina thermophila and Methanoculleus thermophilus

The cofactor F420 is synthesized by many different organisms and as a redox cofactor, it plays a crucial role in the redox reactions of catabolic and biosynthetic metabolic pathways. It consists of a deazaflavin structure, which is linked via lactate to an oligoglutamate chain, that can vary in length. In the present study, the methanogenic Archaea Methanosarcina thermophila and Methanoculleus thermophilus were cultivated on different carbon sources and their coenzyme F420 composition has been assayed by reversed-phase ion-pair high-performance liquid chromatography with fluorometric detection regarding both, overall cofactor F420 production and distribution of F420 glutamyl tail length. In Methanosarcina thermophila cultivated on methanol, acetate, and a mixture of acetate and methanol, the most abundant cofactors were F420-5 and F420-4, whereby the last digit refers to the number of expressed glutamyl rests. By contrast, in the obligate CO2 reducing Methanoculleus thermophilus the most abundant cofactors were F420-3 and F420-4. In Methanosarcina thermophila, the relative proportions of the expressed F420 tail length changed during batch growth on all three carbon sources. Over time F420-3 and F420-4 decreased while F420-5 and F420-6 increased in their relative proportion in comparison to total F420 content. In contrast, in Methanoculleus thermophilus the relative abundance of the different F420 cofactors remained stable. It was also possible to differentiate the two methanogenic Archaea based on the glutamyl tail length of the cofactor F420. The cofactor F420-5 in concentrations >2% could only be assigned to Methanosarcina thermophila. In all four variants a trend for a positive correlation between the DNA concentration and the total concentration of the cofactor could be shown. Except for the variant Methanosarcinathermophila with acetate as sole carbon source the same could be shown between the concentration of the mcrA gene copy number and the total concentration of the cofactor.

Quantification of Histidine-Containing Dipeptides in Dolphin Serum Using a Reversed-Phase Ion-Pair High-Performance Liquid Chromatography Method

The quantification of histidine-containing dipeptides (anserine, carnosine, and balenine) in serum might be a diagnostic tool to assess the health condition of animals. In this study, an existing reversed-phase ion-pair high-performance liquid chromatography (HPLC)–ultraviolet detection method was improved and validated to quantify serum anserine, carnosine, and balenine levels in the dolphin. The serum was deproteinized with trichloroacetic acid and directly injected into the HPLC system. Chromatographic separation of the three histidine-containing dipeptides was achieved on a TSK–gel ODS-80Ts (4.6 mm × 150 mm, 5 µm) analytical column using a mobile phase of 50 mmol/L potassium dihydrogen phosphate (pH 3.4) containing 6 mmol/L 1-heptanesulfonic acid and acetonitrile (96:4). The standard curve ranged from 0.1 µmol/L to 250 µmol/L. The average accuracy of the intra- and inter-analysis of anserine, carnosine, and balenine was 97–106%. The relative standard deviations of total precision (RSDr) of anserine, carnosine, and balenine in dolphin serum were 5.9%, 4.1%, and 2.6%, respectively. The lower limit of quantification of these compounds was 0.11–0.21 µmol/L. These results indicate that the improved method is reliable and concise for the simultaneous determination of anserine, carnosine, and balenine in dolphin serum, and may be useful for evaluation of health conditions in dolphins. Furthermore, this method can also be applied to other biological samples.

Isolation and Characterization of Nitrate Reducing Bacteria for Conversion of Vegetable-Derived Nitrate to ‘Natural Nitrite’

In the US, sodium nitrate is used as a preservative and curing agent in processed meats and is therefore a regulated ingredient. Nitrate reducing bacteria (NRB) can convert vegetable nitrate into nitrite allowing green/clean label status in the US as per the USDA-FSIS definition of ‘natural nitrite’. The current ‘in-liquid’ test tube assay for detecting nitrite is not suitable for screening mixtures of bacteria nor is commercial nitrate broth suitable for growth of many Gram (+) bacteria. M17 broth was therefore used to develop M17-nitrate broth to be inclusive of Gram (+) bacteria. An ‘on-agar’ colony-screening assay was developed to detect the conversion of nitrate to nitrite on agar plates and could detect one NRB+ colony among ~300–500 colonies on a single plate. Samples that might have NRB were spread-plated on M17 agar plates, sandwiched with nitrate agar, and after incubation followed with sequential agar overlays containing the reagents used in the nitrate reduction assay; the appearance of red color zones above colonies indicated the presence of nitrite. NRB derived from various samples were confirmed for nitrate conversion and both nitrate and nitrite were quantified by C8 reversed-phase (RP) ion-pairing high performance liquid chromatography (HPLC) analysis (1 ppm limit of detection). Staphylococcus carnosus, a strain commonly used for nitrate reduction, was able to convert 1100 ppm M17-nitrate broth to 917 ppm nitrite. Staphylococcus caprae and Panteoa agglomerans, NRB isolated using the M17-nitrate agar assay, were also able to ferment the same broth to 916 ppm and 867 ppm nitrite, respectively. This is the first report of an on-agar colony screening assay for the detection and isolation of nitrite reducing bacteria allowing NRB to be readily isolated. This may allow for the identification of new bacteria that may have a more efficient process to generate nitrite, and possibly concomitant with production of additional natural antimicrobials, as vegetable nitrite becomes more widely used to prevent spore germination.

Selenium Species Determination in Se-Enriched Grain Crops with Foliar Spray of Sodium Selenite by IP-RP-HPLC-UV-HG-AFS

A simple, rapid, and accurate method for determination of selenocystine (SeCys2), methyl selenocysteine (MeSeCys), selenourea (SeUr), selenite (Se(IV)), selenotmethionine (SeMet), and selenate (Se(VI)) in grain crops was developed, based on the coupling of ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) and ultraviolet irradiation hydride generation atomic fluorescence spectrometry (UV-HG-AFS). The conditions of IP-RP-HPLC-UV-HG -AFS were studied. The conditions of the pretreatment sample, such as types and dosage of proteinases, were investigated. The detection limits (LODs) for six Se species were between 0.77 and 1.77 μg·L−1. Then, the proposed method has been successfully used to analyze selenium (Se) species in Se-enriched rice paddy (Oryza sativa L.), soybean (Glycine max (Linn.) Merr.), and sweet potato (Ipomoea batatas (L.) Lam.) with foliar application of sodium selenite solution. SeMet was the major Se spices in Se-enriched grain crops, ranged from 77–90% of the total Se contents. The capabilities of Se enrichment were relatively larger differences in three-grain corps as follows: soybean > rice paddy > sweet potato.

Determination of trigonelline in human plasma by magnetic solid-phase extraction: a pharmacokinetic study.

Aim: To develop a novel method for the bioanalytical extraction of trigonelline (TRG) from human plasma samples using a magnetic nanocomposite and to evaluate its pharmacokinetic profile. Materials & methods: Magnetic bentonite/β-cyclodextrine (β-CD) nanoparticles, coupled with a validated ion-pairing reversed-phase high-performance liquid chromatographymethod, were used to determine TRG concentration from plasma samples following a single oral administration. Results: The developed reversed-phase high-performance liquid chromatography method was accurate, precise, specific, selective and reproducible. TRG showed rapid absorption, middle rate of elimination and mean residence time of ∼24h. The data were best fitted on a two-compartment model in which tmax was 1.0h, Cmax 0.115μg/ml, area under the curve (AUC)0-24 1.72μg/ml.h, Cl 0.0293 l/h/kg, t1/2α 0.79h, t1/2β 13.68h and ka 1.63h-1. Conclusion: The findings of this study could provide useful information to promote the future study of TRG and aid optimal dose finding.

Brain/blood ratios of methadone and ABCB1 polymorphisms in methadone-related deaths

Methadone is an opioid that often leads to fatalities. Interpretation of toxicological findings can be challenging if no further information about the case history is available. The aims of this study were (1) to determine whether brain/blood ratios can assist in the interpretation of methadone findings in fatalities; (2) to examine whether polymorphisms in the gene encoding the P-glycoprotein (also known as multidrug resistance protein 1 (MDR1) or ATP-binding cassette sub-family B member 1 (ABCB1)), which functions as a multispecific efflux pump in the blood–brain barrier, affect brain/blood ratios of methadone. Femoral venous blood and brain tissue (medulla oblongata and cerebellum) from 107 methadone-related deaths were analysed for methadone by gas chromatography-mass spectrometry. In addition, all the samples were genotyped for three common ABCB1 single nucleotide polymorphisms (SNPs rs1045642, rs1128503, and rs2032582) using ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry (ICEMS). In nearly all cases, methadone concentrations were higher in the brain than in the blood. Inter-individual brain/blood ratios varied (0.6–11.6); the mean ratio was 2.85 (standard deviation 1.83, median 2.35). Moreover, significant differences in mean brain/blood ratios were detected among the synonymous genotypes of rs1045642 in ABCB1 (p = 0.001). Cases with the T/T genotype had significantly higher brain/blood ratios than cases with the other genotypes (T/T vs. T/C difference (d) = 1.54, 95% CI [1.14, 2.05], p = 0.002; T/T vs. C/C d = 1.60, 95% CI [1.13, 2.29], p = 0.004). Our results suggest that the rs1045642 polymorphisms in ABCB1 may affect methadone concentrations in the brain and its site of action and may be an additional factor influencing methadone toxicity.

Simultaneous Determination of Five Whitening Agents by Ion-Pair Reversed-Phase High Performance Liquid Chro- matography

ABSTRACT A gradient ion-air reversed-phase high performance liquid chromatography method was developed for simultaneous determination of 5 whitening agents (ascorbic acid (AA), magnesium ascorbyl phosphate (MAP), kojic acid (KA), arbutin (ART) and hydroquinone (HQ)). The gradient elution was performed using Inertsil ODS-3V column (250 × 4.6 mm, 5 μm). The analytes were detected by ultraviolet light absorption at the wavelength of 270 nm, the flow rate was set at 1.0 mL/min, and the sample injection volume was 10 μL. The buffer solution contains 50 mM sodium phosphate monobasic dihydrate and 2 mM n-hexadecyltrimethyl ammonium bromide at pH 4.5. The precision of the proposed method, expressed as relative standard deviation (RSD) (%), was determined by the analysis of each compound. The RSD range was from 0.23 to 0.55%. The calibration curves were found to be linear in 8.12-40.6 μg/mL (KA), 12.44-62.20 μg/mL (ART), 8.28-41.40 μg/mL (HQ), 8.16-40.80 μg/mL (MAP) and 4.25- 1.80 μg/mL (AA). The correlation coefficient of linear regression analysis (R2) was within the range of 0.9968 - 0.9997. The analytical results were satisfied for these 5 whitening agents. The recoveries ranging from 96.97% to 101.22% for lotion, and from 97.17 to 100.80% for cream, were obtained at both low and high concentrations. The proposed method is suitable for routine analysis of the 5 whitening agents in commercial cosmetic products, or examined the allowable amount of whitening agents for the Department of Health.

Quantitative assessments of adenosine triphosphatase hydrolytic activity by ultrafiltration-coupled ion-pair reversed-phase high-performance liquid chromatography.

Adenosine triphosphate is a universal energy currency that can directly provide energy required for a multitude of biochemical reactions and biophysical actions through adenosine triphosphatase catalyzed hydrolysis. Adenosine triphosphatase activity is thus one important feature for the characterization of protein function and cell activity. Herein, we optimized ion-pair reversed-phase high-performance liquid chromatography technique for highly efficient separation of adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate, and the method demonstrated good linearity. Moreover, by coupling a protein-removable ultrafiltration, we developed a sensitive and robust approach for quantification of adenosine triphosphatase hydrolytic activity. By this assay, we demonstrated that RecA filaments-catalyzed adenosine triphosphate hydrolysis approached a second-order reaction, and its rate constant was estimated as 0.057mM-1 min-1 . In addition, we explored the effects of DNA length on this reaction and revealed that the increase of the length of single-stranded DNA can promote the adenosine triphosphatase hydrolytic activity of RecA filaments. All these results confirm the feasibility of this new method in quantification of adenosine triphosphatase hydrolytic activity assays. Compared with previous complicated enzyme-coupled or homogeneous colorimetric measurements, the developed approach with high resolution separation allows a simple reaction system for adenosine triphosphatase assay and a sensitive detection free of interference from background noise.

Development and validation of an ion-pair HPLC chromatography for simultaneous determination of lactone and carboxylate forms of SN-38 in nanoparticles

A simple and reproducible reversed-phase ion-pair high performance liquid chromatography (HPLC) method using isocratic elution with UV absorbance detection was developed and validated for the simultaneous determination of carboxylate (C) and lactone (L) forms of SN-38 (active metabolite of irinotecan) in nanoparticles (NPs). Reversed phase (RP) chromatography was performed on a C18 column with an ion-pair solution, KH2PO4 buffer solution and acetonitrile, under a mobile phase 30:30:40 (v/v) and at a flow rate of 1 mL/min. Detection was performed at 265 nm and sharp peaks were obtained for C and L forms of SN-38 at retention times of 2.9 ± 0.01 and 5.1 ± 0.01 min respectively. Linear regression analysis data for the calibration plot showed a good linear relationship between response and concentration in the range of 4 to 100 μg/mL; the regression coefficient was 0.9999 for L form and 0.9998 for C form. The optimized method showed good performance in terms of specificity, linearity, detection and quantitation limits, precision and accuracy in accordance with the International Conference on Harmonization (ICH) Q2 (R1) guidelines. This assay was demonstrated to be applicable for routine quantitation of SN-38 in PLGA NPs.

Simultaneous determination of bromhexine HCl and baicalin in Chinese compound medicine by a reversed-phase ion-pair HPLC

In this article, a reversed-phase ion-pair high-performance liquid chromatography (HPLC) method was established for simultaneous separation and quantitative analysis of bromhexine HCl (BHH) and baicalin in Chinese Ke-Chuan-Ling compound capsule. The influence of the mobile phase composition on the retention of the analytes was investigated. The optimal determination conditions consisted of acetonitrile/water/phosphoric acid/triethylamine (22/78/0.1/0.1, v/v/v/v) mobile phase with the addition of tetra-n-butylammonium bromide (TBA) (0.7 mmol L-1, terminal concentration) as pairing ion, and a detection wavelength at 225 nm. In this case, baicalin and bromhexine HCl were eluted at 21 min and 27 min respectively and were separated successfully from other ingredients. Excellent linear relationship between the peak area and concentration was obtained in the range of 0.2-15.0 μg mL-1 for bromhexine HCl and 0.1-12.0 μg mL-1 for baicalin. The limit of detection of BHH was 0.1 μg mL-1 and it was 0.05 μg mL-1 for baicalin. The average recoveries of bromhexine HCl and baicalin were 97.7% and 99.1%, respectively. From the results it can be concluded that this method is simple, versatile, and reliable for the quality control of Chinese traditional medicines containing baicalin and/or bromhexine HCl.

A Rapid Method for Analysis of cDNA Synthesis Using Ion-Pair Reversed-Phase High Performance Liquid Chromatography

We have developed a rapid, quantitative method for analysing the outcome of the first strand synthesis step in cDNA library preparation, yield and molecular weight range of the final cDNA products are determined after size fractionation. This method involves conventional cDNA library construction including all enzymatic steps usually required, but replaces radioactive labelling of nucleic acids with fluorescence detection. The separation and quantification steps all involve ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC). This quantitative method replaces the use of autoradiography and size exclusion chromatography with combined ion-pair reversed-phase high performance liquid chromatography and in line fluorescence detection. The result of this approach is combination of speed with the generation of reproducible, high quality cDNA libraries.

Impurity profiling of l-aspartic acid and glycine using high-performance liquid chromatography coupled with charged aerosol and ultraviolet detection

For the compendial related substances test of l-aspartic acid (Asp) and glycine (Gly), two separate reversed-phase ion-pair high-performance liquid chromatography methods coupled with charged aerosol and ultraviolet detection were developed. Separation of all putative impurities, in particular of the related carboxylic and amino acids, was achieved using volatile perfluorocarboxylic acids as ion-pairing reagents on a polar embedded C18 stationary phase. It was shown that an adjustment of the evaporation temperature of the charged aerosol detector (CAD) was an efficient strategy for meeting the required quantitation limits, when dealing with non-volatile analytes. It was also demonstrated that the usage of a two detector setup can be beneficial for extending the detection range and providing accurate quantitation of low level impurities (LOQs from 5 to 50 ng on column). Both methods were validated with accordance to ICH guideline Q2(R1) assessing specificity, linearity, accuracy, precision, and robustness. Several batches of Asp and Gly were tested for related substances using the developed methods. The purity of each sample was higher than 99.7 %. Coupled charged aerosol and UV detection proved to be a more simple, robust and selective alternative to established derivatization procedures such as the Amino-Acid-Analyser (AAA) for the impurity profiling of amino acids, and should thus be considered for implementation into pharmacopoeial monographs in the future.

Simultaneous Determination of Cysteamine and Cystamine in Cosmetics by Ion-Pairing Reversed-Phase High-Performance Liquid Chromatography

Cysteamine has been used in cosmetics as an antioxidant, a hair straightening agent, and a hair waving agent. However, recent studies indicate that cysteamine can act as an allergen to hairdressers. The objective of this study was to develop and validate a simple and effective reversed-phase high-performance liquid chromatography (RP-HPLC) method for the measurement of cysteamine and its dimer, cystamine. Sodium 1-heptanesulfonate (NaH-pSO) was used as an ion-pairing agent to improve chromatographic performance. Separation was performed on a Gemini C18 column (250 mm × 4.6 mm, 5 µm particle size) using a mobile phase composed of 85:15 (v/v) 4 mM NaHpSO in 0.1% phosphoric acid:acetonitrile. UV absorbance was monitored at 215 nm. The RP-HPLC method developed in this study was validated for specificity, linearity, limit of detection, limit of quantitation, precision, accuracy, and recovery. Cysteamine and cystamine were chromatographically resolved from other reducing agents such as thioglycolic acid and cysteine. Extraction using water and chloroform resulted in the recovery for cysteamine and cystamine ranging from 100.2–102.7% and 90.6–98.7%, respectively. This validated RP-HPLC method would be useful for quality control and monitoring of cysteamine and cystamine in cosmetics.

Targeted Determination of Tissue Energy Status by LC-MS/MS.

Intracellular nucleotides and acyl-CoAs are metabolites that are central to the regulation of energy metabolism. They set the cellular energy charge and redox state, act as allosteric regulators, modulate signaling and transcription factors, and thermodynamically activate substrates for oxidation or biosynthesis. Unfortunately, no method exists to simultaneously quantify these biomolecules in tissue extracts. A simple method was developed using ion-pairing reversed-phase high-performance liquid chromatography–electrospray-ionization tandem mass spectrometry (HPLC-ESI-MS/MS) to simultaneously quantify adenine nucleotides (AMP, ADP, and ATP), pyridine dinucleotides (NAD+ and NADH), and short-chain acyl-CoAs (acetyl, malonyl, succinyl, and propionyl). Quantitative analysis of these molecules in mouse liver was achieved using stable-isotope-labeled internal standards. The method was extensively validated by determining the linearity, accuracy, repeatability, and assay stability. Biological responsiveness was confirmed in assays of liver tissue with variable durations of ischemia, which had substantial effects on tissue energy charge and redox state. We conclude that the method provides a simple, fast, and reliable approach to the simultaneous analysis of nucleotides and short-chain acyl-CoAs.

"Determination of nitrate and nitrite content in Zonar milk serum and derived dairy drinks using ion-pair reversed-phase high performance liquid chromatography "

"Determination of nitrate and nitrite content in Zonar milk serum and derived dairy drinks using ion-pair reversed-phase high performance liquid chromatography "

HPLC–UV method for simultaneous determination of adenosine triphosphate and its metabolites in Mycobacterium smegmatis

Method for the analysis of intracellular adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) in Mycobacterium smegmatis that involves rapid extraction procedure based on sonication of cells in perchloric acid, as well as separation of nucleotides by ion-pair reversed-phase high-performance liquid chromatography and ultraviolet (UV) detection at 254 nm, is developed. The analytes were separated with mobile phase consisted of acetonitrile and 50 mM monobasic potassium phosphate (pH 4.6) with 25 mM tetrabutylammonium hydrogensulfate in a ratio of 0.5:99.5% within 30 min. The calibration curves were linear in the range of 20–1000 pmol of ATP and 10–1000 pmol of ADP and AMP with correlation coefficient (r2) of ≥0.9998. The proposed method is applicable for mycobacterium cultures taken over a wide range of optical density and physiological states. Concentrations of ATP, ADP, and AMP in mycobacterial extracts varied from 2.61 ± 0.27 to 9.60 ± 0.19 nmol/mg dry weight, from 1.75 ± 0.12 to 5.86 ± 0.09 nmol/mg dry weight, and from 0.55 ± 0.08 to 4.40 ± 0.07 nmol/mg dry weight, respectively, depending on the physiological state.