Strong Cation Exchange Column Research Articles

Development of chromatographic methods to determine multivitamins formulation depending on their solubility and polarity: comparative study using three greenness assessment tools

High performance liquid chromatography is one of the techniques of choice for the separation and quantitative determination of drugs in mixture form. Ipriflavone, ascorbic acid, pyridoxine, vitamin D3, and lysine are formulated together as an adjuvant combination in osteoporosis. In this work, we developed and validated two complementary high performance liquid chromatographic methods to determine the five compounds in their pharmaceutical dosage form. The first method (method A) was capable of determining ipriflavone, ascorbic acid, pyridoxine, and vitamin D3 in their bulk and combined pharmaceutical formulation. The method is based on Liquid Chromatographic separation with UV detection at 254 nm using Agilent Eclipse XDB-C18 column with a mobile phase consisting of 25 mM ammonium acetate buffer (pH 4.2): methanol in gradient mode. Due to the high polarity of lysine, it was difficult to achieve satisfactory retention on reversed phase columns. So, we separated it on a strong cation exchange column (Exsil 100 SCX) without derivatization with a mobile phase consisting of 10 mM sodium dihydrogen phosphate and 200 mM sodium chloride (pH 6) with UV detection at 210 nm (method B). Validation of the proposed methods was performed according to ICH guidelines Q2(R1). The proposed methods proved to be valid for selective analysis of the stated drugs in their bulk and combined pharmaceutical formulation. Greenness assessment of the developed methods was evaluated using three assessment tools: ESA, GAPI and the most recently developed tool AGREE, showing a satisfactory comprehensive guide of the greenness of the developed methods.

A Differential Protein Study on Bronchoalveolar Lavage Fluid at Different Stages of Silicosis.

In this study, by comparing the difference in protein expression in bronchoalveolar lavage fluid between silicosis patients in different stages and healthy controls, the pathogenesis of pneumoconiosis was discussed, and a new idea for the prevention and treatment of pneumoconiosis was provided. The lung lavage fluid was pretreated by 10 K ultrafiltration tube, Agilent 1100 conventional liquid phase separation, strong cation exchange column (SCX) HPLC pre-separation, and C18 reverse phase chromatography desalting purification, and protein was labeled with isotope. GO, KEGG pathway, and PPI analysis of differential proteins were conducted by bioinformatics, and protein types and corresponding signal pathways were obtained. Thermo Q-Exactive mass spectrometry identified 943 proteins. T-test analysis was used to evaluate the different significance of the results, and the different protein of each group was obtained by screening with the Ratio≥1.2 or Ratio≤0.83 and P<0.05. We found that there are 16 kinds of protein throughout the process of silicosis. There are different expressions of protein in stages Ⅲ/control, stages Ⅱ/control, stage Ⅰ/control, stages Ⅲ/ stages Ⅱ, stages Ⅲ/ stage Ⅰ and stages Ⅱ/ stage Ⅰ groups. The results of ontology enrichment analysis of total differential protein genes show that KEGG pathway enrichment analysis of differential protein suggested that there were nine pathways related to silicosis. The main biological changes in the early stage of silicosis are glycolysis or gluconeogenesis, autoimmunity, carbon metabolism, phagocytosis, etc., and microfibril-associated glycoprotein 4 may be involved in the early stage of silicosis. The main biological changes in the late stage of silicosis are autoimmunity, intercellular adhesion, etc. Calcium hippocampus binding protein may participate in the biological changes in the late stage of silicosis. It provides a new idea to understand the pathogenesis of silicosis and also raises new questions for follow-up research.

An enzymatic fluorimetric assay for determination of N-acetylaspartate

N-acetylaspartate (NAA) is an abundant metabolite in the mammalian brain and a precursor of the neuropeptide N-acetylaspartylglutamate (NAAG). The physiological role of NAA is not fully understood and requires further studies. We here describe the development of a coupled enzymatic fluorimetric assay for the determination of NAA in biological samples. Deproteinized tissue extracts are first passed through a strong cation exchange column to remove aspartate. NAA in the sample is hydrolysed by aspartoacylase and released aspartate oxidized using l-aspartate oxidase. Generated H2O2 is measured with peroxidase in a fluorimetric assay using Ampliflu Red. The limit of detection and the lower limit of quantification are 1.0 μM (10 pmol/well) and 3.3 μM (33 pmol/well), respectively, with a linear range to 100 μM. Specificity of the assay was confirmed using samples from mice deficient in NAA synthase Nat8l that were spiked with NAA. Analysis of samples from aspartoacylase-deficient mice showed a 2 to 3-fold increase in brain NAA concentration, in line with previous reports. Mice lacking NAAG synthetases had a slightly reduced (−10%) brain NAA level. Thus, the new fluorimetric enzymatic assay is useful to perform sensitive and large scale quantification of NAA in biological samples without the need for expensive equipment.

Development and Validation of a Strong Cation Exchange Chromatographic Column Coupled with High-Performance Liquid Chromatography Method for Meropenem and Evaluation of Its Stability in Human Plasma: Application to the Therapeutic Drug Monitoring.

Meropenem is a wide inter-individual variability in the pharmacokinetic, and standard dosing may not be adequate in critically ill patients. Therapeutic drug monitoring is a useful tool to optimize dosing. Meropenem is the amphoteric compound with an isoelectric point of 5.15. The secondary amino group of meropenem is positively charged when pH ≤ 5.4, thus we attempted to separate by strong cation exchange (SCX) column using acetonitrile/25-mM potassium dihydrogen phosphate (pH3.0; 60:40) as mobile phase, and good peak shape and effective separation obtained. Generally, meropenem were unstable in plasma. We try to investigate stability of plasma samples using the medium QC sample with or without 3-(N-morpholino) propanesulfonic acid (MOPS) as stabilizer solutions at possible conditions during handling and storage. Meropenem showed higher stability at -80°C, and addition of MOPS might increase the short-term and extracted samples stability. This method is suitable for the quantification of meropenem in human plasma from 0.5 to 100μg/mL. The accuracy was ranged from 96.53 to 101.11% with relative standard deviation ≤ 4.76%. The method has been used for determined 63 critically ill patients treated with meropenem. During the first measurement, 11 patients showed trough levels below the target ranges despite standard dosing. Through continuous or prolonged infusion, 8/11 patients (72.73%) led to adequate trough levels. The described SCX-high-performance liquid chromatography method for meropenem in human plasma is a powerful tool for therapeutic drug monitoring.

Optimized Chromatographic Determination of Naphazoline and Chlorpheniramine in Eye/Nose Drops Using Cation-exchange Column.

A simple and efficient liquid chromatographic method has been developed and validated for the simultaneous determination of naphazoline and chlorpheniramine in eye/nose drops, in presence of naphazoline degradation product and naphthalene acetic acid (NAPD). The separation was achieved within 6.0min, employing a mixture of 53.5% v/v water-acetonitrile containing 78.70mM/L sodium dihydrogen orthophosphate adjusted to pH5.30 as isocratic mobile phase, pumped at 1.0mL/min through a strong cation-exchange column (10μm particle size), the analytes were monitored at 230nm. Statistical experimental designs and graphic representations (response surface methodology) were used for optimizing the chromatographic separation. The linearity plots were linear over concentration range up to 125% of the analytes nominal concentrations (100%) with regression coefficients (r) > 0.99, method's accuracy (RSD < 2.0%), repeatability and intermediate precision (RSD < 2.0%) were verified. System suitability parameters were also within the acceptable range. The validated method was successfully employed for the routine analysis of eye/nose drops.

基于集成化多柱二维液相色谱系统分析血清中的氨磺必利

快速准确的治疗药物监测对于临床上确保患者用药有效性及安全性至关重要,同时也能够确定患者用药依从性,制定个性化给药方案。该文以两支疏水性略有差异的反相分离柱Supersil ODS2和SinoChrom ODS-BP,及强阳离子交换捕集柱Supersil SCX构建了基于集成化的多柱二维液相色谱系统。通过二维色谱接口,以pH 3.0的磷酸缓冲液调整第一维分离后的洗脱液组成,降低有机相含量并维持pH,改善了中心切割模式下样品转移和捕集的效率。利用该多柱二维液相色谱系统发展了血清中氨磺必利的二维液相色谱检测方法,血清样品经过高氯酸和甲醇混合液沉淀蛋白质并离心后直接300 μL大体积进样,以乙腈/磷酸缓冲液(25 mmol/L, pH 3.0)(20/80, v/v)作为第一维分离流动相,磷酸缓冲液(25 mmol/L, pH 3.0)作为捕集过程的稀释流动相,乙腈/磷酸缓冲液(25 mmol/L, pH 7.0)(25/75, v/v)作为第二维分离流动相,12 min内即可完成分析。方法在10~200 ng/mL的范围内线性相关性良好(r=0.9998)。样品在50 ng/mL和100 ng/mL两个加标浓度下的回收率稳定,在73.7%~76.8%之间。方法的检出限为7.28 ng/mL,定量限为24.27 ng/mL,能够满足《神经精神药理学治疗药物检测共识指南》中推荐的药物监控范围要求。由于该系统日常使用及维护成本较低,且能够实现自动化分析,故该方法适合在临床上用于治疗药物监测研究。

Extraction and detection of quaternary ammonium ionic liquid cations in water samples

The recent development of ionic liquids may induce environmental pollution, yet there is a lack of sensitive analytical methods to detect traces of ionic liquids, notably for quaternary ammonium ionic liquids. Here, we set up a method based on solid-phase extraction and ion chromatography combined to analyze quaternary ammonium ionic liquid cations in river water samples. Cations included tetramethylammonium, tetraethylammonium, tetrapropylammonium and tetrabutylammonium. We used methanesulfonic acid–acetonitrile as mobile phase in a cation-exchange column with carboxylic acid functional groups. A strong acid cation-exchange solid-phase extraction column was used for extraction and enrichment, then water–methanol was eluted to wash impurities, and finally the analytes were eluted with H3PO4–KH2PO4 buffer solution because this buffer has a low conductivity signal which avoids interferences with cation detection. We obtained a detection limit of 0.003 mg/L for river samples. Precision and accuracy met the requirements of quantitative analysis.

Separation and screening of antioxidant peptides from Scomberomorus niphonius based on nano flow liquid chromatography

As a rich source of high activity antioxidant peptides, Scomberomorus niphonius is a key marine natural product with a high processing value. Due to the high complexity of fish tissues, high recovery extraction and high efficiency screening of the active antioxidant peptide species have become the major challenges for the research and development of preparation and separation techniques. Due to the high specificity of hydrolytic enzymes, when different types of enzymes are used for the hydrolysis of fish tissues, the resultant active peptides may have significant differences in chemical structures, biological functions, and physical activities. In this study, in order to obtain antioxidant peptides with high activity and functionality, defatted visceral powder of Scomberomorus niphonius was used as a raw material for sample preparation. Five hydrolytic enzymes (flavor protease, trypsin, acid protease, neutral protease, and alkaline protease) were selected and investigated for their hydrolyzing efficiencies in visceral solutions of Scomberomorus niphonius, according to their optimum hydrolyzing conditions. The diphenyl bitter hydrazine radical (DPPH·) scavenging rate, hydroxyl radical (·OH) scavenging rate, and degree of hydrolysis (DH) were adopted as indicators for hydrolytic enzyme selection and optimization. The data suggested that, among all the hydrolytic enzymes investigated, trypsin presented the best scavenging capabilities for both DPPH· and ·OH species, with scavenging rates of 88.93%±0.82% for DPPH· and 53.09%±0.73% for ·OH, respectively. Based on the single-factor test results, the DPPH· scavenging rate was further utilized as an indicator and was found to depend on the hydrolytic enzyme quantity, hydrolyzation temperature, and hydrolyzation time. According to the Box-Behnken center composed experimental design, a triple factor and triple level response surface method was adopted for further optimization of antioxidant peptide preparation from Scomberomorus niphonius viscera. The preparation method was systematically optimized, and as a result, a degree of hydrolysis of 23.66%, DPPH· scavenging rate of 93.78%, and ·OH scavenging rate of 62.59% were achieved. High performance nano flow liquid chromatography is a new generation micro scale liquid phase separation technique that has the advantages of low sample requirement, low solvent consumption, and high efficiency. In this study, we applied this method to marine natural product purification. In order to screen the most suitable chromatographic stationary phase for the separation and analysis of active antioxidant peptides from Scomberomorus niphonius viscera, a nano flow reversed-phase C18 column (15 cm×100 μm, 5 μm, 30 nm) and a strong cation exchange column (15 cm×100 μm, 5 μm, 100 nm) were investigated. A nano flow high performance liquid chromatography platform with a 1:1000 splitting ratio was adopted for this study. Using the enzyme-hydrolyzed solution from Scomberomorus niphonius viscera as the sample, following filtration, the antioxidant peptides were separated, collected, freeze-dried, and finally tested for their antioxidant capability. The results showed that the strong cation exchange phase was more suitable for antioxidant peptide isolation and purification from Scomberomorus niphonius viscera, and an antioxidant peptide component with high activity was successfully screened. During the activity test, the 50% inhibition concentration (IC50) value of the DPPH· scavenging capability of this peptide was 0.672±0.051 mg/mL, which was 13.6 times higher than the activity value before the peptide species was purified. The current study for the first time reports the application of high performance nano flow liquid chromatography in the purification and analysis of antioxidant peptides from a marine natural product source and also demonstrates the effectiveness and future prospect of the use of nano flow ion exchange chromatography for high performance separation and high efficiency screening of antioxidant peptides.

Column Classification/Characterisation of Strong Cation Exchange Phases for the Liquid Chromatographic Analysis of Small Molecular Weight Bases

A simple, rapid and robust protocol for the characterisation of strong cation exchange columns for the analysis of small molecular weight bases is described. A range of ten different phases were characterised, and the resultant selectivity and retention factors analysed using Principal Component Analysis. The score plots for the first and second principal components described 83% of the variability within the dataset. Score plots highlighted the large chromatographic differences observed between the phases, the validity of which was established using a larger range of bases. All the strong cation exchange materials demonstrated a synergistic mixed mode (i.e. ion exchange and hydrophobic) retention mechanism. Principal Component Analysis also highlighted the potential difficulty in locating suitable strong cation exchange “back-up” columns for the analysis of small molecular weight bases in that the characterised columns all displayed very different selectivities. The robustness of the protocol was confirmed by a factorial design experiment.

Determination of cobalamin in chlorella food by cation exchange column and graphite furnace atomic absorption spectrometry

A method for the rapid determination of cobalamin (Vitamin B12) in chlorella food was proposed. This method utilized a strong cation exchange column to remove free cobalt (II), and the cobalamins were quantitatively determined by measuring total cobalt in the column effluent with graphite furnace atomic absorption spectrometry (GFAAS). Under optimized conditions, the method detection limit was 24 μg/100 g. The method was validated by spiking cobalamin standards in different chlorella food, recoveries from spike preparations ranged from 98.3 to 99.5%, with coefficients of variation of 1.2-4.9%. The results show the suitability of the proposed method for determining vitamin B12 in chlorella food.

Determination of Risperidone and 9-Hydroxyrisperidone in Human Serum by Heart-Cutting Isocratic Two-Dimensional Liquid Chromatography

This study reports a novel heart-cutting two-dimensional high performance liquid chromatography (2D-LC) method with ultraviolet detection for the determination of risperidone and 9-hydroxyrisperidone in human serum. In this 2D system, a strong cation-exchange column was applied for the first dimensional (1D) separation to isolate the targets from the complex serum matrices, and a middle column was used to transfer the targets from the first dimension to the second dimension (2D) through a six-port valve. A reverse-phase C18 column was selected in the second dimension for further separation. An assistant pump was optimized to prevent the peak dispersion and the loss of resolution. This method has the advantages of high sensitivity by large injection volume, simple one-step sample preparation and short analysis time. A linear range between 5 and 146 ng/mL for risperidone and 7 and 200 ng/mL for 9-hydroxyrisperidone was achieved. All of the results showed that the coupling of ion-exchange with reversed-phase liquid chromatography had powerful separation capabilities for risperidone and 9-hydroxyrisperidone in human serum. The method exhibited good repeatability and recoveries and may be used as a reliable approach for therapeutic drug monitoring in clinical analysis.

Development of an on-line immunoextraction/entrapment system for protein capture and use in drug binding studies by high-performance affinity chromatography

An on-line purification and entrapment system was developed that could extract a protein from a sample such as serum and entrap this protein within a small column for use in high-performance affinity chromatography. Human serum albumin (HSA) was employed as a model protein for this work. Immunoextraction columns containing polyclonal anti-HSA antibodies were developed to capture and isolate HSA from applied samples. This was followed by the use of a strong cation-exchange column to recapture and focus HSA as it eluted from the immunoextraction columns. The recaptured HSA was entrapped within 1.0 cm × 2.1 mm I.D. columns containing hydrazide-activated silica and in the presence of oxidized glycogen as a capping agent. The binding and elution properties of HSA on the various components of this system were examined and optimized. The entrapped columns produced by this system were then evaluated for their use in binding studies with several sulfonylurea drugs. The HSA columns created by this approach typically contained 0.3–0.6 nmol HSA and were stable over several weeks and more than 50–60 sample injections. Drug binding constants could be determined with these columns in 8 min or less by zonal elution and gave good agreement with literature values. The same system could be used for the capture and entrapment of other proteins by utilizing antibodies against the given target for immunoextraction.

Ion exchange in supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS): Application for polar and ionic drugs and metabolites in forensic and anti-doping analysis

Supercritical fluid chromatography (SFC) is an orthogonal technique to UHPLC. In recent years, SFC has demonstrated potential for use in the analysis of a broad variety of analytes of different polarities, if modifiers and additives are utilized as additional mobile phase constituents. However, to date, little research has been carried out on ion-exchange separation of highly polar and ionic analytes using SFC.The objective of this work was to investigate the elution characteristics of polar compounds using SFC combined with tandem mass spectrometry. Highly polar and even ionic drugs and metabolites, with a diversity of functional groups such as gamma-hydroxybutyrate (GHB), gamma-butyrolactone, GHB-glucuronide, ethyl sulfate, ethyl glucuronide as well as meldonium and gamma-butyrobetaine, were selected for the study. To investigate the chromatographic behavior of the solutes using SFC, a systematic chromatographic method development workflow including a basic validation in human urine was implemented. To ensure the best selectivity, columns with different stationary phase chemistries (silica, NH2, CN, SCX, Diol, EP and amide) were screened. Furthermore, different modifier compositions were evaluated, including pure methanol and methanol with various additives (ammonium acetate or ammonium formate, water, and/or ammonia or formic acid in varying amounts). Trends in retention time shifts were investigated by the systematic variation of gradients, backpressure and column compartment temperature. The quality and reliability of the method has been tested in the course of a basic validation by evaluation of selectivity, linearity, limit of identification, limit of quantification, carry-over, precision, and matrix effect.The highest chromatographic selectivity (especially with respect to the separation of the critical pair of zwitterionic meldonium and γ-butyrobetaine) and suitable peak shapes for all target analytes were obtained using the strong cation exchange column. We observed that increasing buffer concentrations improved the peak shape. Ion-exchange stationary phase and polar additives (buffers, bases, acids and water) mixed with organic modifiers enabled separation of small ionic molecules using SFC. Hence, SFC further demonstrated its wide applicability in small molecule analysis, and may be considered an alternative separation technique to improve the separation of polar analytes.

Preparative separation of isoquinoline alkaloids from Corydalis impatiens using a middle-pressure chromatogram isolated gel column coupled with two-dimensional liquid chromatography.

We established a two-dimensional strong cation exchange/reversed-phase liquid chromatography protocol to isolate and purify isoquinoline alkaloids from Corydalis impatiens. Isoquinoline alkaloids were first enriched from a C. impatiens extract in which liposoluble components were removed using a medium-pressure chromatographic tower containing middle chromatogram isolated gel. A strong cation exchange column was employed to separate and obtain 30 fractions. We chose fractions 22-29 for reversed-phase liquid chromatography purification using characteristic isoquinoline alkaloid ultraviolet absorption spectra. Several isoquinoline alkaloid fractions (22-29) were further separated, and those of low resolution were isolated via two-dimensional liquid chromatography in the orthogonal plane. A total of eight novel isoquinoline alkaloids with characteristic ultraviolet spectra were obtained from C. impatiens. We thus demonstrate the benefits of off-line two-dimensional strong cation exchange/reversed-phase liquid chromatography to isolate isoquinoline alkaloids from C. impatiens.

Purification of tertiary and quaternary alkaloids from Rhizoma Corydalis using reversed-phase/weak cation-exchange mixed-mode class separation combined with preparative C18 and silica based strong cation-exchange chromatography

A new optimization strategy for purification of alkaloids from Rhizoma Corydalis using preparative liquid chromatography was developed, featuring a selective separation of different types of alkaloids into different parts by a reversed-phase/weak cation-exchange mixed-mode column (named C18WCX) at first. The total alkaloids of Rhizoma Corydalis were divided into four fractions with fraction III and IV corresponding to the tertiary type medium bases and the quaternary type strong bases, respectively. For fraction III, a conventional C18 column was used to isolate tertiary alkaloids using acetonitrile and 0.1% phosphoric acid (adjusted with triethylamine to pH 6.0) as mobile phases. High selectivity and symmetrical peak shapes of tertiary alkaloids were obtained, resulting in six main tertiary alkaloids isolated in a single run. As strong bases, quaternary alkaloids often suffer from serious peak tailing problem on conventional C18 columns. Therefore, a silica-based strong cation-exchange (SCX) column was used for purification of fraction IV. On the SCX column, good peak shapes in high sample loading were achieved. Five main quaternary alkaloids were isolated and identified from the fraction in one-step. The procedures presented effective for the preparative isolation and purification of alkaloids from Rhizoma Corydalis.

A method for the mg scale separation of curium(III) from americium(III) by HPLC using a SCX column

A method intended for the mg scale separation of curium (Cm) from americium (Am) and plutonium (Pu) based on high pressure liquid chromatography using a strong cation exchange column has been investigated. Reproducible separations were achieved on the Luna SCX (Phenomenex) column with α-hydroxymethylbutyric acid (α-HMBA) as eluant. The separation performance was investigated on the tracer level varying the pH, concentration, temperature and flow rate of the eluting solution. It was found that 0.1 M α-HMBA at a pH of 3.7 using a flow-rate of 1 mL/min, gave enough peak resolution for an efficient and reproducible separation of Cm from Am. Separations with gadolinium loading were investigated to scale up the purification of Cm from tracer level to mg scale. Baseline chromatographic separations were demonstrated in column loadings of up to 20% of full column capacity, corresponding to 2.5 mg of Cm.

Synthesis of (R,S)-isoproterenol, an inhibitor of tau aggregation, as an 11C-labeled PET tracer via reductive alkylation of (R,S)-norepinephrine with [2-11C]acetone.

(R,S)-Isoproterenol inhibits the formation of toxic granular tau oligomers associated with neuronal loss and development of cognitive disorders, and is an attractive drug candidate for Alzheimer's disease. To elucidate its behavior in the brain by positron emission tomography, we synthesize (R,S)-[11C]isoproterenol by reductive alkylation of (R,S)-norepinephrine with [2-11C]acetone, which was in turn synthesized in situ under improved conditions afforded a decay-corrected radiochemical yield of 54%. The reductive alkylation using NaBH(OAc)3 as reducing agent in the presence of benzoic acid in DMSO/DMF (60:40 v/v) at 100 °C for 10 min gave (R,S)-[11C]isoproterenol in an 87% radio-high performance liquid chromatography (HPLC) analytical yield. HPLC separation using a strong cation exchange column, followed by pharmaceutical formulation in the presence of d/l-tartaric acid, afforded (R,S)-[11C]isoproterenol with a total radioactivity of 2.0 ± 0.2 GBq, a decay-corrected radiochemical yield of 19 ± 2%, chemical and radiochemical purities of 71% and >99%, respectively, and a molar activity of 100 ± 13 GBq/μmol (n = 3). The overall synthesis time from the end of the bombardment to pharmaceutical formulation was 48 min. A preliminary preclinical PET study in a rat demonstrated the potential of the radioligand for the evaluation of the penetration of (R,S)-isoproterenol in human brain.

Direct Analysis of Underivatized Amino Acids in Plant Extracts by LC-MS/MS (Improved Method).

In this chapter we describe a method for quantification of 20 proteinogenic amino acids by liquid chromatography-mass spectrometry which affords neither derivatization nor the use of organic solvents. Analysis of the underivatized amino acids is performed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) in the positive ESI mode. Separation is achieved on a strong cation exchange (SCX) column (Luna 5μ SCX 100Å) with 5% acetic acid in water (A) and 75mM ammonium acetate in water (B). Quantification is accomplished by use of d2-phenylalanine as internal standard achieving limits of detection of 5-50nM. The method was successfully applied for the determination of proteinogenic amino acids in plant extracts.

Universal retention standard for peptide separations using various modes of high-performance liquid chromatography

Peptide retention standards are widely used by chromatography specialists. They can be used for quality control of peptide separations (separation efficiency, selectivity, retention values) and for accurate concatenation of retention data from multiple acquisitions in proteomics. So far the repertoire of available retention standards is mostly limited to reversed-phase separations. We introduce a synthetic peptide mixture which can be used in conjunction with the most popular peptide separation techniques: reversed-phase (RPLC), strong-cation exchange (SCX), (strong-anion exchange) SAX and hydrophilic interaction liquid chromatography (HILIC). Target sequences were first designed in-silico using Sequence-Specific Retention Calculator models covering all major peptide separation mechanisms. Peptides were also designed while keeping in mind the simplicity of retention time assignment using MS detection: they all have nearly identical masses and identical intense y3 fragment ions. This contribution demonstrates the application of this mixture for characterization of eight HILIC as well as SAX, SCX and C18 columns.

A simplified, rapid LC-MS/MS assay for serum and salivary creatinine

In routine clinical laboratories, serum creatinine is typically measured on automated analyzers using colorimetric or enzymatic assays, which are both susceptible to interferences that can lead to incorrect measurement. Here, we present a straightforward and rapid LC-MS/MS assay for serum creatinine using methanol extraction, with separation performed using a strong cation exchange column. Results from this newly developed method were compared against those from an automated Abbott Architect kinetic Jaffe method. We also assessed the effect of bilirubin and glucose, as interferants, on both methods.Our LC-MS/MS assay has a run time of 1.1 min, uses a relatively small sample volume of 10 µL and has a within-batch imprecision of 1.1–1.8% at the concentrations tested, which is within the range necessary for routine clinical use. Recovery from serum samples spiked with creatinine was >95%, and glucose and bilirubin were not found to interfere in the assay. Glucose was observed to significantly interfere in the kinetic Jaffe method, while bilirubin had a variable effect. We also determined that creatinine can be measured from saliva using our method, and that salivary concentrations are, on average, 15% of those in serum. This assay provides an alternative for patient sample analysis where interference is expected in routine creatinine methods.