Ion-pair High-performance Liquid Chromatography Research Articles

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.

Quantification of aromatic amines derived from azo colorants in textile by ion-pairing liquid chromatography tandem mass spectrometry

Azo dyes can metabolize back to precursor aromatic amines (arylamines), which are potentially carcinogenic. The ISO 14362–1:2017 standard requires the determination of the arylamines, produced from textile samples, preferably by using the gas chromatography mass spectrometric (GC-MS) method. This paper presents an ion-pairing high performance liquid chromatography tandem mass spectrometric (LC-MS/MS) method for determining aromatic amines, derived from azo colorants, in both natural and synthetic textiles. The separation enables adequate apparent retention factor (k’ > 2.1 for the most hydrophilic compounds) and has appropriate sensitivity without sample clean-up procedure. The background matrix constituents influence the quantification in even a 100-fold diluted sample, therefore, calibration requires background compensation. The method allows fast confirmation and quantification of twenty-five arylamines in complex matrices of textiles. The method was validated with success and used to both natural and synthetic textile proficiency test (PT) samples.

Quantitative Analysis of Glycine Oligomerization by Ion-Pair Chromatography.

This paper describes a method for the quantitative analysis of mixtures of glycine and its oligomers by ion-pair high-performance liquid chromatography (IP-HPLC), with a particular focus on applications in origins-of-life research. We demonstrate the identification of glycine oligomers (Glyn) up to 14 residues long—the approximate detectable limit of their solubility in water—and measurement of the concentration of these species in the product mixture of an oligomerization reaction. The molar response factors for higher oligomers of glycine—which are impractical to obtain as pure samples—are extrapolated from direct analysis of pure standards of n = 3–6, which established a clear linear trend. We compare and contrast our method to those in previous reports with respect to accuracy and practicality. While the data reported here are specific to the analysis of oligomers of glycine, the approach should be applicable to the design of methods for the analysis of oligomerization of other amino acids.

A Comparative HPLC Analysis of Myricetin, Quercetin and Kaempferol Flavonoids Isolated From Gambian and Indian Moringa oleifera Leaves

Moringa oleifera has been used for centuries as a traditional medicine in a number of sub-tropical countries. Recent studies have found it to be rich in flavonoids that exhibit antioxidant activity both in vitro and in vivo. In this study slightly modified conditions were examined in order to maximise the yield of these compounds in particular; Myricetin, Quercetin and Kaempferol. Quantitative analysis of these flavonoids were established using reverse phase ion-pairing High Performance Liquid Chromatography (HPLC) at a wavelength of 370 nm. The method development was carried out following the International Conference for Harmonization guidelines. It was found that refluxing with 0.10 M hydrochloric acid for 24 hour provided high yields of the flavonols (myricetin 292 mg/kg, quercetin 1099 mg/kg and kaempferol 133 mg/kg). There was a significant difference in the yield of these flavonols originating from the different geographical locations, with Community Forest Management Farm (CFM Farm) yielding the highest quantity of the flavonols under the conditions applied.

Quantitative analysis of underivatized amino acids in the sub- to several-nanomolar range by ion-pair HPLC using a corona-charged aerosol detector (HPLC–CAD)

The separation and quantification of underivatized naturally occurring amino acids (AAs) by high-performance liquid chromatography (HPLC) is advantageous for reducing preparation time, running costs, and analytical errors, and is compatible with the isolation of intact AAs. This study establishes and validates an analytical method for the separation and quantification of underivatized AAs and taurine (Tau) in the sub- to several-nanomolar range (ca. 0.1–1.6 nmol) by optimizing ion-pair HPLC coupled to a corona charged aerosol detector (corona CAD). Chromatographic separation of 19 AAs and Tau was achieved using a porous graphite carbon (PGC) column and nonafluoropentanoic acid (NFPA) as a volatile ion-pair reagent. The response of the corona CAD to the AAs was highly dependent on the eluate composition, whereas these response factors were similar for AAs in eluate with similar compositions. Regression curves and coefficients (r2) >0.998 were obtained for plots of injection amount versus peak area, except for Arg which co-eluted with a background peak. On the other hand, all plots of injection amount versus peak height regressed to curves with r2 > 0.997. Repeat quantification based on peak area showed lower relative standard deviations (RSDs) (typically better than 5%) than those based on peak height. The present method is useful for quantifying AAs from proteins and Tau in the sub- to several-nanomolar range without derivatization, and constant repeatability can be maintained by quantification using peak areas.

Biomethylation metabolism study of arsenite in SCC-7 cells by reversed phase ion pair high performance liquid chromatography-inductively coupled plasma-mass spectrometry

Arsenite (As(III)) has been considered as a human carcinogen associated with many human cancers especially skin cancer. Elucidation of the transformed species of As(III) during its metabolism in cells is beneficial for evaluation of its bioeffect. In this work, a hyphenated method of reversed phase ion pair high performance liquid chromatography - inductively coupled plasma mass spectrometry (RP-IP-HPLC-ICP-MS) equipped with collision/reaction cell technology (CCT) was developed for speciation of As(III) and its metabolites (arsenate [As(V)], monomethylarsonic acid [MMA(V)], and dimethylarsinic acid [DMA(V)]) in SCC-7 cells. The developed analytical method exhibits low limits of detection for interest arsenic species in the range of 14–27 ng/L and wide linear range up to four orders of magnitude, providing a sensitive tool for arsenic metabolites analysis and further understanding the metabolism of As(III) in SCC-7 cells. The effect of exposure time, exposure concentrations and elimination time on the arsenic species and total arsenic in SCC-7 cells incubated by As(III) were systematically studied. At low exposure concentrations (< 5 μM), large proportion of intracellular As(III) transformed to methylated metabolites, and the final methylated metabolite DMA(V), which could not be completely removed from the cells in the elimination process, is considered to play as the primary carcinogen. While at high exposure concentrations (> 5 μM), most of intracellular As(III) probably bound to biomacromolecules rather than followed biomethylation process, exhibiting different metabolism.

Simultaneous Determination of Succinic Acid and Water-Soluble Vitamins by Ion-Pair High-Performance Liquid Chromatography

A procedure is proposed for the determination of succinic acid, riboxin, nicotinamide, and riboflavin by ion-pair HPLC with UV detection. Owing to the addition of an ion-pair modifier to the mobile phase and the selection of gradient elution conditions, the optimal retention and resolution of peaks of the components to be determined are achieved. Specificity, linearity, accuracy, and precision of the developed procedure are proved on an example of the determination of active substances in the Cerebronorm® preparation.

Bile acid patterns in commercially available oxgall powders used for the evaluation of the bile tolerance ability of potential probiotics.

This study aimed to analyze the bile acid patterns in commercially available oxgall powders used for evaluation of the bile tolerance ability of probiotic bacteria. Qxgall powders purchased from Sigma-Aldrich, Oxoid and BD Difco were dissolved in distilled water, and analyzed. Conjugated bile acids were profiled by ion-pair high-performance liquid chromatography (HPLC), free bile acids were detected as their p-bromophenacyl ester derivatives using reversed-phase HPLC after extraction with acetic ether, and total bile acids were analyzed by enzymatic-colorimetric assay. The results showed that 9 individual bile acids (i.e., taurocholic acid, glycocholic acid, taurodeoxycholic acid, glycodeoxycholic acid, taurochenodeoxycholic acid, glycochenodeoxycholic acid, cholic acid, chenodeoxycholic acid, deoxycholic acid) were present in each of the oxgall powders tested. The content of total bile acid among the three oxgall powders was similar; however, the relative contents of the individual bile acids among these oxgall powders were significantly different (P < 0.001). The oxgall powder from Sigma-Aldrich was closer to human bile in the ratios of glycine-conjugated bile acids to taurine-conjugated bile acids, dihydroxy bile acids to trihydroxy bile acids, and free bile acids to conjugated bile acids than the other powders were. It was concluded that the oxgall powder from Sigma-Aldrich should be used instead of those from Oxoid and BD Difco to evaluate the bile tolerance ability of probiotic bacteria as human bile model.

Construction of an optimized method for quality evaluation and species discrimination of Coptidis Rhizoma by ion-pair high performance liquid chromatography combined with response surface methodology

Coptidis Rhizoma (CR), the dried rhizome of three perennial Coptis specices, was widely used as a famous herbal medicine in China. Although the quantification of main alkaloids in CR has been extensively conducted, the existing analytical methods suffer from some flaws that restrict the general applicability in the routine quality assessment. In this work, we constructed an optimized method for quality evaluation and species discrimination of CR by ion-pairing high performance liquid chromatography (IP-HPLC) combined with response surface methodology (RSM). By employing Box-Behnken designs (BBD), 30 sets of experimental runs were performed to build the response surface models, and Derringer’s desirability was used to optimize the IP-HPLC separation conditions by simultaneously taking resolutions between two pairs of hardly − separated alkaloids and the retention time of the last eluted analyte as optimization criteria. Meanwhile, a single standard to determine multi-components (SSDMC) method based on the optimized IP-HPLC was set up and fully validated, to simultaneously determine six alkaloids including jatrorrhizine (JAT), columbamine (COL), epiberberine (EPI), coptisine (COP), palmatine (PAL) and berberine (BER), using BER as internal standard. Finally, the quantitative data from 33 batches of CR samples were comparatively analyzed, and the ratios of JAT/COL and EPI/JAT were discovered for species classification. Collectively, the established IP-HPLC method can be adopted for comprehensive quality evaluation and species discrimination of CR due to its general applicability.

Degradation of Reactive Black 5 by electrochemical oxidation

Degradation of commercial grade Reactive Black 5 (RB5) azo dye by chemical and electrochemical treatment was examined using a dimensionally stable anode and stainless steel cathodes as electrode materials, with NaCl as supporting electrolyte. The electrochemical treatment was compared to the chemical treatment with hypochlorite generated by electrolysis. The compounds present in the commercial grade RB5 azo dye and the products of its electrochemical degradation were separated using ion-pairing high performance liquid chromatography on reversed phase. The separated species were detected by diode array detector and electrospray ionization mass spectrometry. A suitable ion-pairing reversed phase HPLC-MS method with electrospray ionization for the separation and identification of the components was developed. The accurate mass of the parent and fragment ions were used in the determination of the empirical formulas of the components using the first-order mass spectra. Structural formulas of degradation products were proposed using these information and principles of organic chemistry and electrochemistry.

Determination of Fluoroquinolones in Animal Feed by Ion Pair High-performance Liquid Chromatography with Fluorescence Detection

ABSTRACTA sensitive and simple method is described for the determination of fluoroquinolones by high-performance liquid chromatography (HPLC) using a C18 column and fluorescence detection. The mobile phase was acetonitrile and 0.025M phosphate acid with 0.0025M sodium 1-heptanesulfonate monohydrate in water with a gradient program. The chromatographic conditions were optimized for the determination of ciprofloxacin, enrofloxacin, sarafloxacin, and flumequine in animal feed. The samples were extracted by a hydrochloric acid and acetonitrile followed by dilution in 0.01M oxalic acid at pH 4.0 and purified by solid-phase extraction. The procedure was validated by fortifying feed at 200, 1000, and 2000 µg/kg. The limits of detection and quantification were from 28.5 to 74.7 and 31.7 to 94.6 µg/kg, respectively. The average recoveries for fluoroquinolones were from 89.7 to 100.3%. The method was validated and shown to be efficient and precise for quantification of fluoroquinolones in animal feed. The results demonstrate the feasibility of the method for routine use to monitor these substances in feed.

First report of cyanobacterial paralytic shellfish toxin biosynthesis genes and paralytic shellfish toxin production in Polish freshwater lakes

&lt;p&gt;In central and southern Europe, &lt;em&gt;Aphanizomenon&lt;/em&gt; spp., &lt;em&gt;A. gracile&lt;/em&gt; Lemmermann in particular, have been associated with paralytic shellfish toxin (PST) production. In western Poland, &lt;em&gt;A. gracile&lt;/em&gt; is very common, and &lt;em&gt;Cylindrospermopsis raciborskii&lt;/em&gt; (Woloszyńska) Seenayya &amp;amp; Subba Raju, another potentially PST-producing species, is often found as well. To date it is, however, unknown if the cyanobacterial populations in this area harbour the genetic capability to produce PSTs, and to what extent toxin biosynthesis occurs. The objective of this study was to survey the prevalence of potentially PST-producing cyanobacteria by measuring paralytic shellfish toxin biosynthesis gene &lt;em&gt;sxtB&lt;/em&gt; copy numbers, &lt;em&gt;sxtA&lt;/em&gt;, &lt;em&gt;sxtG&lt;/em&gt; and &lt;em&gt;sxtS&lt;/em&gt; gene presence, and PST concentrations in Polish freshwater lakes. In total, 34 lakes in western Poland were sampled twice during summer 2010. The presence of PST biosynthesis genes &lt;em&gt;sxtA&lt;/em&gt;, &lt;em&gt;sxtG&lt;/em&gt; and &lt;em&gt;sxtS&lt;/em&gt; was determined using conventional qualitative PCR. Quantitative PCR (qPCR) was used to measure &lt;em&gt;sxtB &lt;/em&gt;copy numbers, and the samples were analysed for PSTs using ion-pair high performance liquid chromatography with post-column oxidation and fluorescence detection (HPLC-FLD). Cyanobacteria carrying the &lt;em&gt;sxtB&lt;/em&gt; gene were present in 23.5% of all samples (n=16) and in 14 lakes of the studied 34. Gene copy numbers ranged from 8.2×10&lt;sup&gt;4&lt;/sup&gt; to 5.1×10&lt;sup&gt;7&lt;/sup&gt; &lt;em&gt;sxtB&lt;/em&gt; copies L&lt;sup&gt;-1&lt;/sup&gt; (mean 3.8×10&lt;sup&gt;6&lt;/sup&gt;). The median was 4.5×10&lt;sup&gt;5&lt;/sup&gt; &lt;em&gt;sxtB&lt;/em&gt; gene copies L&lt;sup&gt;-1&lt;/sup&gt; and the majority of results clustered at the lower end of the &lt;em&gt;sxtB&lt;/em&gt; qPCR linear range. In 12 out of the 16 samples positive for &lt;em&gt;sxtB&lt;/em&gt; the gene co-occurred with the other three targeted PST biosynthesis genes &lt;em&gt;sxtA&lt;/em&gt;, &lt;em&gt;sxtG&lt;/em&gt; and &lt;em&gt;sxtS&lt;/em&gt;. However, five additional samples lacked one or two of the targeted four genes. Thirteen samples contained PSTs, of which 12 samples at levels &amp;lt;0.072 µg L&lt;sup&gt;-1&lt;/sup&gt;, &lt;em&gt;i.e.&lt;/em&gt;, close to or below the quantitative detection limit of the HPLC-FLD method (0.01 µg L&lt;sup&gt;-1&lt;/sup&gt;). One sample contained 0.57 µg L&lt;sup&gt;-1&lt;/sup&gt; saxitoxin, co-occurring with all four &lt;em&gt;sxt&lt;/em&gt; genes studied. No correlation between PST and &lt;em&gt;sxt&lt;/em&gt; gene occurrence or copy numbers was observed. &lt;em&gt;A. gracile&lt;/em&gt; and &lt;em&gt;C. raciborskii&lt;/em&gt; occurred in 92% and 50% of samples, respectively, containing PSTs, &lt;em&gt;sxt&lt;/em&gt; genes or both. In conclusion, the results confirm that potential PST producers constitute an established subpopulation of cyanobacteria in Polish freshwater lakes. However, none of the &lt;em&gt;sxt&lt;/em&gt; genes targeted in this study could serve as a reliable marker for active PST biosynthesis.&lt;/p&gt;

Separation of Glutathionate-Protected Gold Clusters by Reversed-Phase Ion-Pair High-Performance Liquid Chromatography

Recent studies have demonstrated that reversed-phase high-performance liquid chromatography (RP-HPLC) is a very effective means of separating thiolate (SR)-protected gold clusters (Aun(SR)m). In the present study, we applied RP-HPLC to the separation of hydrophilic glutathionate (SG)-protected Aun(SG)m clusters. To achieve such separation, an ion pair reagent was dissolved in the solution to form ion pairs with the functional groups on the cluster surfaces, improving the interaction between the cluster surfaces and the hydrophobic stationary phase, representing ion pair chromatography. This technique resulted in the high-resolution separation of Aun(SG)m clusters via RP-HPLC. Experiments at different solution pH values led to slight improvements in the resolution, showing that pH adjustment, which modifies the efficiency of the ion pair formation process, is a useful technique for improving this type of separation.

Reduced molecular size and altered disaccharide composition of cerebral chondroitin sulfate upon Alzheimer's pathogenesis in mice.

Alzheimer's disease (AD) is a progressive disorder leading to cognitive impairment and neuronal loss. Cerebral extracellular accumulation and deposition of amyloid ß plaques is a pathological hallmark of AD. Chondroitin sulfate (CS) is an extracellular component abundant in the brain. CS is a sulfated glycosaminoglycan covalently attached to a core protein, forming chondroitin sulfate proteoglycan. The structure of CS is heterogeneous with sulfation modification and elongation of the chain. The structural diversity of CS allows it to play various roles in the brain. Increasing evidence has shown that CS promotes aggregation of amyloid ß peptides into higher-order species such as insoluble amyloid ß fibrils. Difficulties in the structural analysis of brain CS, as well as its heterogeneity, limit the study of potential roles of CS in AD pathology. Here we established a microanalysis method with reversed-phase ion-pair high performance liquid chromatography and found that CS in the brains of Tg2576 AD model mice show a lower molecular size and an increased ratio of CS-B motif di-sulfated disaccharide. Our findings provide insight into the structural changes of cerebral CS upon Alzheimer's pathogenesis.

Based on Nucleotides Analysis of Tumor Cell Lines to Construct and Validate a Prediction Model of Mechanisms of Chemotherapeutics.

Cancer is one of the diseases that seriously threaten to human life worldwide. Up to now, chemotherapy remains to be a critical means of cancer treatment, thus the development of chemotherapeutical drugs has become a top priority. An ion pair high performance liquid chromatography (ion pair RP-HPLC) was established for analyzing intracellular nucleotides of tumor cell lines. In this article, a partial least-squares discriminant analysis (PLS-DA) prediction model of mechanisms of chemotherapeutics was established based on four types of drugs with different mechanisms, including antimetabolic agents, antineoplastic agents that affect protein synthesis, agents directly acting on DNA, and RNA interference agents. Then four anti-tumor agents commonly used in clinical were used to validate the availability of the prediction model. Three natural compounds, including 16- dehydropregnenolone (16-DHP), apigenin (API) and diosgenin (DIO), were reported to display anti-tumor effect with unclear mechanisms. The three components were applied to this prediction model firstly. In conclusion, the recognition model was proved to be accurate and feasible to some degree and might become a promising auxiliary method in the process of chemotherapeutic drugs development.

Ion-pairing HPLC methods to determine EDTA and DTPA in small molecule and biological pharmaceutical formulations

Ion-pairing high-performance liquid chromatography–ultraviolet (HPLC–UV) methods were developed to determine two commonly used chelating agents, ethylenediaminetetraacetic acid (EDTA) in Abilify® (a small molecule drug with aripiprazole as the active pharmaceutical ingredient) oral solution and diethylenetriaminepentaacetic acid (DTPA) in Yervoy® (a monoclonal antibody drug with ipilimumab as the active pharmaceutical ingredient) intravenous formulation. Since the analytes, EDTA and DTPA, do not contain chromophores, transition metal ions (Cu2+, Fe3+) which generate highly stable metallocomplexes with the chelating agents were added into the sample preparation to enhance UV detection. The use of metallocomplexes with ion-pairing chromatography provides the ability to achieve the desired sensitivity and selectivity in the development of the method. Specifically, the sample preparation involving metallocomplex formation allowed sensitive UV detection. Copper was utilized for the determination of EDTA and iron was utilized for the determination of DTPA. In the case of EDTA, a gradient mobile phase separated the components of the formulation from the analyte. In the method for DTPA, the active drug substance, ipilimumab, was eluted in the void. In addition, the optimization of the concentration of the ion-pairing reagent was discussed as a means of enhancing the retention of the aminopolycarboxylic acids (APCAs) including EDTA and DTPA and the specificity of the method. The analytical method development was designed based on the chromatographic properties of the analytes, the nature of the sample matrix and the intended purpose of the method. Validation data were presented for the two methods. Finally, both methods were successfully utilized in determining the fate of the chelates.

Ion-pair HPLC method for the quantification of metformin in human urine

Metformin is an oral antidiabetic drug that has been used in the treatment of non-insulin-dependent diabetes, which improves glycemic control by primarily inhibiting hepatic gluconeogenesis and glucogenolysis [1]. Metformin is slowly absorbed after oral administration, about 60% of an oral dose is excreted in the urine as unchanged drug within 24 h, and about 30% of the dose is nonabsorbed and eliminated unchanged in feces [2]. Previous studies have shown a plasma elimination half-life ranging from 2.0 to 6.0 h after oral administration of varying doses (0.5 to 1.5 g/dose) [3]. Urine provides a non-invasive sample collection method, and determination of drug levels in urine is comparatively less complex than plasma and other body fluids [4]. Several reports indicate that urinary excretion data can be used to arrive at bioequivalence decision of different drug formulations [4-6]. Chemically, it is 1,1-dimethyl biguanide (Figure 1). Metformin is a small highly polar molecule (pKa=2.8, 11.5, log P octanol:water= −2.6), which has great solubility in water and poor solubility in lipids; it is also possible to retain in reversed-phase (RP) columns using ion-pairing reagents. Numerous methods for the quantitation of metformin in urine have been utilized over years. They include capillary electrophoresis with contactless conductivity detection [7], voltammetric method [8], cation exchange high-performance liquid chromatography (HPLC) [9], reverse phase RP-HPLC [10-12] and liquid chromatography (LC)–mass spectrometry (MS) [13]. Most of the methods have been based on HPLC with spectrophotometric detection in the range of 230–240 nm (Table 1). Chemical derivatization of metformin has been used JOURNAL OF APPLIED BIOANALYSIS, January 2016, p. 16-24. http://dx.doi.org/10.17145/jab.16.004 (ISSN 2405-710X) Vol. 2, No. 1

Reversed‐phase ion‐pair high‐performance liquid chromatography assay of polyprenyl diphosphate oligomer homologues

A reversed-phase ion-pair high-performance liquid chromatography procedure was developed for the separation of polyprenyl diphosphate oligomer homologues obtained chemically from plant polyprenols. Tetrabutylammonium phosphate was used as the ion-pair reagent, and the dependence of the separation quality on pH of ion-pair reagent was investigated for the first time. The procedure is applicable for the control of commercial available polyprenyl monophosphates (the active components of veterinary drugs Phosprenyl and Gamapren) for the possible presence of polyprenyl diphosphate byproducts.

Determination of β-agonists in Porcine Meats by Ion-Pair Extraction and High Performance Liquid Chromatography

ABSTRACTIon-paired extraction was employed for the determination of salbutamol, clenbuterol, and ractopamine. Tetrabutylammonium bromide was used as the ion-pairing agent and water–acetonitrile was the extraction solvent. The concentration and pH of the buffer, salt addition, concentration of tetrabutylammonium bromide, and type and volume of extraction solvent were optimized. No centrifugation step was required. Matrix interferences from real samples were eliminated using matrix-matched calibration. Under the optimum conditions, a linear calibration relationship was present from 0.0025 to 3 µg mL−1 with an enrichment factor of more than 25. This method was combined with high-performance liquid chromatography for the determination of β-agonists in porcine meats with low limits of detection, high recovery, and rapid analysis.

Three-phase hollow-fiber microextraction combined with ion-pair high-performance liquid chromatography for the simultaneous determination of five components of compound α-ketoacid tablets in human urine.

The determination of α-ketoacid concentration is demanded to evaluate the absorption and metabolic behavior of compound α-ketoacid tablets taken by chronic kidney disease patients. To eliminate the interference of endogenous substance of urine and enrich the analytes, a three-phase hollow-fiber liquid-phase microextraction combined with ion-pair high-performance liquid chromatography method was established for the determination of d,l-α-hydroxymethionine calcium, d,l-α-ketoisoleucine calcium, α-ketovaline calcium, α-ketoleucine calcium, and α-ketophenylalanine calcium of compound α-ketoacid tablets in human urine samples. The extraction parameters, such as organic solvent, pH of donor phase and acceptor phase, stirring rate, and extraction time were optimized. Under the optimal conditions, the obtained enrichment factors were up to 11-, 110-, 198-, 202-, and 50-fold, respectively. The calibration curves for these analytes were linear over the range of 0.1-10 mg/L for α-ketovaline calcium, d,l-α-ketoisoleucine calcium, and α-ketoleucine calcium, 0.5-10 mg/L for d,l-α-hydroxymethionine calcium, and α-ketophenylalanine calcium with r > 0.99. The relative standard deviations (n = 5) were less than 6.27% and the LODs were 100.7, 10.0, 5.8, 7.8, and 8.6 μg/L (based on S/N = 3), respectively. Good recoveries from spiked urine samples (92-118%) were obtained. The proposed method demonstrated excellent sample clean-up and analytes enrichment to determine the five components in human urine.