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

Nanoscale Ion-Pair Reversed-Phase HPLC−MS for Sensitive Metabolome Analysis

In this article, we introduce a method using nanoscale ion-pair reversed-phase high-performance liquid chromatography (nano-IP-RP-HPLC) hyphenated to nanoelectrospray ionization high-resolution mass spectrometry (nano-ESI-HRMS) to separate and identify metabolites in cell extracts. Separation of metabolites was performed on a 100 μm i.d. C18 column with tributylamine (TBA) as the ion-pairing reagent and methanol as the eluent. Basic pH (9.4) of the mobile phase was critical to achieve sufficient retention and sharp metabolite elution at a low concentration of TBA (1.7 mM). Limits of detection were determined for 54 standards with an LTQ-Orbitrap mass spectrometer to be in the upper attomole to low femtomole range for key metabolites such as nucleotides, phosphorylated sugars, organic acids, and coenzyme A thioesters in solvent as well as in a complex matrix. To further evaluate the method, metabolome analysis was performed injecting different amounts of biomass of the methylotroph model organism Methylobacterium extorquens AM1. A (12)C/(13)C labeling strategy was implemented to improve metabolite identification. Analysis of three biological replicates performed with 1.5 ng of cell dry weight biomass equivalents resulted in the identification of 20 ± 4 metabolites, and analysis of 150 ng allowed identifying 157 ± 5 metabolites from a large spectrum of metabolite classes.

Abstract P4-02-17: Polymerase Chain Reaction-Liquid Chromatography-Mass Spectrometry — A Convenient and Cost-Effective Strategy for CYP2D6 Genotyping

Abstract Background: Genetic polymorphisms in the drug metabolizing enzyme Cyotchrome P450 2D6 (CYP2D6) gene can significantly influence a person's ability to mediate CYP2D6 dependent metabolism. For example, a notable influence of the CYP2D6 genotype has been described regarding the conversion of tamoxifen to its pharmacologically active metabolites. Thus, the genetic determination of the CYP2D6 metabolizer status has the potential to represent a valuable therapeutic supplement in breast cancer therapy. However, the clinical relevance of the CYP2D6 genotype regarding the treatment of breast cancer with tamoxifen still remains to be elucidated. Due to the high number of relevant polymorphisms to be screened, pharmacogenetic CYP2D6 testing is considered to be time-consuming and laborious and is therefore limited to a few clinical studies only. To enable a more widespread use, we present polymerase chain reaction (PCR) - ion-pair reversed-phase high-performance liquid chromatography — electrospray ionization time of flight mass spectrometry (ICEMS) as fast, convenient and cost-effective strategy for CYP2D6 genotyping. Material and Methods: After DNA extraction from saliva swabs, the CYP2D6 gene was amplified by a long range PCR. Subsequently, polymorphism-specific short amplicons were generated in a multiplexed PCR. The amplicons were directly analysed by ICEMS. The information necessary to determine the allelic state of a PCR-amplified polymorphic locus were obtained from the measured molecular masses. Gene duplications or deletions were analysed by a long range PCR protocol and subsequent agarose gel detection. Results: The developed CYP2D6 genotyping approach enables the reliable determination of the most frequent CYP2D6 alleles in Europe (*1, *2, *3, *4, *6, *7, *8, *9, *10, *11, *12, *17, *41) including whole gene duplications and deletions (*5). The assay involves the following steps: (1) collecting biological material in form of non-invasive saliva swabs; (2) DNA extraction; (3) long range PCR for a specific CYP2D6 amplification; (3) multiplexed PCR for the generation of polymorphism-specific short amplicons; (4) direct analysis of the reaction mixture with ICEMS; (4) determination of the allelic state with the measured molecular masses; (5) analysis of gene duplications or deletions by a long range PCR protocol and subsequent agarose gel detection. Genotyping of a single sample costs about 5-10 USD and can be accomplished within 2-3 days. Parallelization increases the sample throughput and reduces costs. For proof of principle, we have applied the developed method in the context of the PRO-BETh study to determine the CYP2D6 status of 106 breast cancer patients treated with tamoxifen. According to the classification of Gaedik A. et al [1], 19% of the typed breast cancer patients turned out to be “Poor Metabolizers", 51% were “Intermediate Metabolizers", 29% were “Extensive Metabolizers” and 7% turned out to be “Ultrarapid Metabolizers”. Conclusion: PCR-ICEMS represents a cost-effective and convenient tool for pharmacogenetic testing, which enables the determination of the CYP2D6 status using DNA obtained from a non-invasive saliva swab as template. [1] Gaedigk A. et al, J. Clin Pharmacol Ther. 2007 Jun;81(6):817-20. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-02-17.

Ionic liquids improved reversed-phase HPLC on-line coupled with ICP-MS for selenium speciation

Room-temperature ionic liquids (RTILs) improved reversed-phase high performance liquid chromatography (RP-HPLC) on-line combined with inductively coupled plasma mass spectrometry (ICP-MS) was developed for selenium speciation. The different parameters affecting the retention behaviors of six target selenium species especially the effect of RTILs as mobile phase additives have been studied, it was found that the mobile phase consisting of 0.4% (v/v) 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), 0.4% (v/v) 1-butyl-2,3-dimethylimidazolium tetrafluroborate ([BMMIM]BF 4) and 99.2% (v/v) water has effectively improved the peak profile and six target selenium species including Na 2SeO 3 (Se(IV)), Na 2SeO 4 (Se(VI)), l-selenocystine (SeCys 2), d, l-selenomethionine (SeMet), Se-methylseleno- l-cysteine (MeSeCys), seleno- d, l-ethionine (SeEt) were separated in 8 min. In order to validate the accuracy of the method, a Certified Reference Material of SELM-1 yeast sample was analyzed and the results obtained were in good agreement with the certified values. The developed method was also successfully applied to the speciation of selenium in Se-enriched yeasts and clover. For fresh Se-enriched yeast cells, it was found that the spiked SeCys 2 in living yeast cells could be transformed into SeMet. Compared with other ion-pair RP-HPLC–ICP-MS approaches for selenium speciation, the proposed method possessed the advantages including ability to regulate the retention time of the target selenium species by selecting the suitable RTILs and their concentration, simplicity, rapidness and low injection volume, thus providing wide potential applications for elemental speciation in biological systems.

Heparinase 1 selectivity for the 3,6-di-O-sulfo-2-deoxy-2-sulfamido-α-D-glucopyranose (1,4) 2-O-sulfo-α-L-idopyranosyluronic acid (GlcNS3S6S-IdoA2S) linkages

Porcine intestinal mucosa heparin was partially depolymerized by recombinant heparinase 1 (heparin lyase 1, originating from Flavobacterium heparinum and expressed in Escherichia coli) and then fractionated, leading to the isolation of 22 homogeneous oligosaccharides with sizes ranging from disaccharide to hexadecasaccharide. The purity of these oligosaccharides was determined by gel electrophoresis, strong anion exchange and reversed-phase ion-pairing high-performance liquid chromatography. The molecular mass of oligosaccharides was determined using electrospray ionization-mass spectrometry and their structures were elucidated using one- and two-dimensional nuclear magnetic resonance spectroscopy at 600MHz. Five of the characterized oligosaccharides represent new compounds. The most prominent oligosaccharide comprises the common repeating unit of heparin, ΔUA2S-[-GlcNS6S-IdoA2S-](n)-GlcNS6S, where ΔUA is 4-deoxy-α-l-threo-hex-4-eno-pyranosyluronic acid, GlcN is 2-deoxy-2-amino-d-glucopyranose, IdoA is l-idopyranosyluronic acid, S is sulfate and n=0-7. A second prominent heparin oligosaccharide motif corresponds to ΔUA2S-[GlcNS6S-IdoA2S](n)-GlcNS6S-IdoA-GlcNAc6S-GlcA-GlcNS3S6S (where n=0-5 and GlcA is d-glucopyranosyluronic acid), a fragment of the antithrombin III binding site in heparin. The prominence of this second set of oligosaccharides and the absence of intact antithrombin III binding sites suggest that the -GlcNS3S6S-IdoA2S- linkage is particularly susceptible to heparinase 1.

Evaluation of IP-RP-HPLC for Length Determination of the Trinucleotide Repeat Fragments in Huntington's Disease

Expansion of an unstable trinucleotide (CAG)(n) repeat region within exon 1 of the gene IT15 causes autosomal, dominantly inherited Huntington's disease (HD). The number of CAG-repeats varies from 6 to 35 in normal individuals, whereas in affected patients the expanded allele contains 40 or more CAG-repeats. Thus, exact determination of both alleles of the gene (normal and expanded) on the molecular level is of great importance for clinical diagnosis and prognosis regarding the course of the disease. In our study, we optimized and evaluated a highly sensitive, automated, and economical molecular method for length characterization of the trinucleotide fragment expansion such as (CAG)(n) repeat region based on ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC). We found that IP-RP-HPLC can be used for exact fragment length measuring between 60-280 bp as a sensitive and advantageous alternative method to conventional techniques.

Analysis of Risedronate and Related Substances by Ion-Pair Reversed-Phase High-Performance Liquid Chromatography with Evaporative Light-Scattering Detection

A simple method has been developed for the analysis of risedronate and related substances by ion-pair reversed-phase high-performance liquid chromatography (RPLC) with evaporative light-scattering detection (ELSD). After optimization of the chromatographic conditions, satisfactory separation of the compounds was achieved on an Intersil C(8) column with an isocratic mobile phase: 8:4:88 (v/v) acetonitrile-methanol-12 mM ammonium acetate buffer containing 35 mM n-amylamine (pH 7.0). The mobile-phase flow rate was 1.0 mL min(-1). The calibration plot was linear in the range of 352 to 1760 microg mL(-1) for risedronate. The precision and reproducibility were 0.3 and 0.5%, respectively. The average recovery of risedronate was 100.4% and the RSD was 0.6%. The method was validated and shown to be precise, accurate, and specific for the assay of risedronate in both bulk material and dosage forms. The proposed liquid-chromatographic method can be satisfactorily used for the quality control of risedronate.

Analysis of Fluorescent Dye-labeled Oligonucleotides by Ion-Pair Reversed-Phase High-Performance Liquid Chromatography

Abstract The ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) of fluorescent-labeled oligonucleotides separation was established, and the concentration of triethylamine-acetic acid (TEAA, 0–0.15 M), pH value (4.5–7.0), and gradient strength were optimized. The retention mechanism of fluorescent dye-labeled oligonucleotides was evaluated by comparing the retention of 5, 10, 15-mer unlabeled oligonucleotides with that of 5'end carboxyfluorescein (5'FAM)-labeled oligonucleotides. In addition, TaqMan™ probes as well as other fluorescent dye-labeled oligonucleotides were considered for separation. The results show that the best resolution of different length fluorescent dye-labeled oligonucleotides was observed at a TEAA concentration of 0.01 M and pH 7.0. The retention behavior of fluorescent-labeled oligonucleotides was significantly different from that of the unlabeled isomers; therefore, they can be separated completely. We found that the retention of unlabeled oligonucleotides enhanced with the increase of the length of the molecule. In contrast, the retention of fluorescent-labeled oligonucleotides reduced with the increase of the length of the molecule. Because the hydrophobicity of fluorescent dyes can greatly impact the retention, the oligonucleotides labeled using fluorescent dyes with higher hydrophobicity would have a longer retention time. However, the effect of the hydrophobicity was limited as the length of the oligonucleotide was increased to a certain level.

Fluorometric determination of pantothenic acid in human urine by isocratic reversed-phase ion-pair high-performance liquid chromatography with post-column derivatization

We describe here a method for the determination of pantothenic acid, vitamin B5, in human urine by isocratic reversed-phase ion-pair HPLC with post-column derivatization. Pantothenic acid in urine was separated using a Tosoh ODS-80Ts (4.6 i.d.×250mm) column with phosphate–sodium hydroxide buffer (pH 7.0) containing dodecyltrimethylammonium chloride. Following the isolation of pantothenic acid it was decomposed to pantoic acid and β-alanine by alkali treatment. The product β-alanine was post-derivatized to the fluorescent 1-alkylthio-2-alkylisoindole with orthophthaldialdehyde in the presence of 3-mercaptopropionic acid. In the proposed method, a urine sample can be directly injected into a HPLC system without any pre-clean up treatment. The limit of detection was 3pmol (ca. 650pg) per 20μL of urine at a signal-to-noise ratio of 5:1 and the limit of quantification was 5pmol (ca. 1000pg) per 20μL of urine, which was sufficiently sensitive for the determination of pantothenic acid in human urine. The total time required for the analysis was ca. 25min. The proposed method can be used to assess the pantothenic acid content of human urine as an alternative to the standard microbioassay for pantothenic acid.

Novel approaches for retention time prediction of oligonucleotides in ion-pair reversed-phase high-performance liquid chromatography

The base sequence autocorrelation (BSA) descriptors were used to describe structures of oligonucleotides and to develop accurate quantitative structure-retention relationship (QSRR) models of oligonucleotides in ion-pair reversed-phase high-performance liquid chromatography. Through the combination use of multiple linear regression (MLR) and genetic algorithm (GA), QSRR models were developed at temperatures of 30 °C, 40 °C, 50 °C, 60 °C and 80 °C, respectively. Satisfactory results were obtained for the single-temperature models (STM). Multi-temperature model (MTM) was also developed that can be used for predicting the retention time at any temperature. The correlation coefficients of retention time prediction for the test set based on the MTM model at 30 °C, 40 °C, 50 °C, 60 °C and 80 °C were 0.978, 0.982, 0.989, 0.988 and 0.996, respectively. The corresponding absolute average relative deviations (AARD) for the test set at each temperature were all less than 1%. The new strategy of feature representation and multi-temperatures modeling is a very promising tool for QSRR modeling with good predictive ability for predicting retention time of oligonucleotides at multiple temperatures under the studied condition.

The effect of N‐acetylcysteine on amphetamine‐mediated dopamine release in rat brain striatal slices by ion‐pair reversed‐phase high performance liquid chromatography

The amphetamine (AMPH)-induced alteration in rat brain dopamine levels modified by N-acetylcysteine (NAC) administration has been examined using isocratic ion-pair reversed-phase high-performance liquid chromatography with electrochemical detection. The aim of the development of a novel validated evaluation scheme implying a double AMPH challenge was to enhance the efficiency of AMPH-triggered dopamine release measurements in rat brain striatal slices by improving the reproducibility of the results. The proposed experimental protocol was tested in vivo and proved to be capable of fast and reliable drug screening for tracing the effect of NAC as a model compound in AMPH-mediated dopaminergic response. The subcellular localization of the dopamine mobilizing effect of NAC has been established indirectly by the use of an irreversible dopamine vesicular depletor, reserpine. The antioxidant NAC at 10 mM plays an important role in the complete suppression of acute AMPH-elicited dopamine release. The possible role of this quenching effect is discussed.

An ion-pair reversed-phase HPLC method for determination of fresh tissue adenine nucleotides avoiding freeze–thaw degradation of ATP

Knowledge of the energetic state of tissue is required in a wide range of experimental studies, particularly those investigating the decline and recovery of cellular metabolism after metabolic stress. Such information can be obtained from high-performance liquid chromatography (HPLC) determination of tissue levels of adenine nucleotides (ATP, ADP, and AMP) and their interrelationship in the tissue energy charge (EC). Accordingly, a large range of techniques with which to measure these molecules and their downstream metabolites have been reported. However, the accurate determination of the tissue EC also depends on the nucleotide extraction procedure given that changes in adenine nucleotide levels take place very quickly when ATPases are not inactivated immediately. In this article, we describe an ion-pair reversed-phase HPLC method by which separation of adenine nucleotides can be performed rapidly, allowing multiple analyses in 1 day, with both high sensitivity and extraction efficiency and using fresh samples, thereby avoiding freeze–thaw degradation of nucleotides. We applied this method to hippocampal brain slice extracts and show that same-day extraction and analysis results in a more accurate determination of the in situ energetic state than does the commonly used snap-freezing in liquid nitrogen.

Effective reversed-phase ion pair high-performance liquid chromatography method for the separation and characterization of intact low-molecular-weight heparins

A simple, selective, and efficient reversed-phase ion pair high-performance liquid chromatography (RPIP-HPLC) method was developed for the separation of various commercially available intact low-molecular-weight heparins (LMWHs). The developed method uses a C 18 column (150 × 4.6 mm) with diode array detection at 230 nm, flow rate at 1.0 ml/min, and a mobile phase containing acetonitrile/water (32:68%), tetrabutylammonium hydroxide (15 mM), and ammonium acetate (50 mM) at pH 7.0. The performance of this method was assessed in terms of selectivity, linearity, intra- and interday precision, and accuracy. The novel application of RPIP-HPLC with evaporative light scattering detection (ELSD) for the analysis of intact LMWHs was demonstrated. Intact LMWHs were analyzed with superior resolution and peak shape. Different chromatographic profiles were obtained for different LMWHs showing significant structural diversity. This method clearly showed chemical changes that occurred to LMWH under the stress condition. This method can be applied for the separation, identification, characterization, and pharmaceutical stability analysis of various LMWHs.

On-line separation and identification of inorganic and organic arsenic species in ethanolic kelp and bladderwrack extracts through liquid chromatography/particle beam-electron ionization mass spectrometry (LC/PB-EIMS)

Ion-pair, reversed-phase high-performance liquid chromatography (IP-RP-HPLC) and ion exchange chromatography (IEC), coupled to particle beam-electron ionization mass spectrometry (PB-EIMS), are employed for the separation and identification of inorganic and organic arsenic compounds (As (III) chloride, arsenobetaine (AB), dimethylarsinic acid (DMA) and an arsenosugar (oxo-arsenosugar-glycerol, As 328) in commercial ethanolic kelp and bladderwrack extracts. The reversed-phase separation was performed on a C18 column using an isocratic mobile phase composition of water:methanol (96:4) containing 0.1% of trifluoroacetic acid (TFA) as an ion-pairing agent. The flow rate was optimized at 0.7 mL min−1 and the separation accomplished in less than 8 minutes. Baseline resolution was achieved in an elution window of about 4 minutes. The ion-exchange separation was performed on an anion-exchange column using a gradient mobile phase composition of 0.5 mM nitric acid containing 2% methanol and 50 mM nitric acid. The separation was accomplished in less than 8 minutes at a flow rate of 0.9 mL min−1. The eluted species were detected and identified using a PB-EIMS system providing species-specific information. The influence of various instrument parameters was evaluated to achieve optimal mass spectrometric response for the test compounds. The absolute detection limits of arsenic species with HPLC/PB-EIMS were 0.03, 0.05, 0.008 ng and 0.005 (5 µL injection) in the SIM mode and 0.1, 0.14, 0.04 and 0.01 ng in the TIC mode for As (III), DMA, AB and As 328 respectively. These investigations revealed that the majority (90–95%) of the arsenic present in the ethanolic extract samples is in the form of inorganic arsenic, with minor amounts of As (5–10% of the total As) present in the form of DMA. There were no detectable levels of AB and arsenosugars. The total arsenic content in the ethanolic extracts was verified by ICP-OES and validated by analyzing NIST SRM 3241 Ephedra sinica Stapf Native Extract and SRM 3243 Ephedra-Containing Solid Oral Dosage Form. These investigations indicate that PB-EIMS is a viable approach for comprehensive speciation of various botanical extracts.

The next generation of DNA profiling – STR typing by multiplexed PCR – ion‐pair RP LC–ESI time‐of‐flight MS

For the first time a multiplexed PCR approach suitable for mass spectrometric STR allele identification is presented. Thirteen forensically important STR markers (vWA, D21S11, D3S1358, D16S539, D8S1179, D7S820, D13S317, D5S818, TPOX, CSF1PO, D2S441, D10S1248, and D22S1045) and the gender typing locus amelogenin were simultaneously amplified. Ion-pair reversed-phase high-performance liquid chromatography electrospray-ionization time-of-flight mass spectrometry (ICEMS) was applied for genotyping, and allowed for highly efficient characterization of multiple PCR amplicons. Compared with electrophoretic sizing ICEMS enabled for the simultaneous detection of length and nucleotide variations. Thus, the obtained amount of biological information present within STR profiles was significantly increased even though the compatibility of typing results with electrophoretically generated data(bases) was maintained. Other advantages of the ICEMS platform included the abandonment of internal size standards, allelic ladders, and any kind of spectral calibration. The 14-plex PCR was tailor-made for ICEMS analysis by designing primer pairs that bind close to the repeat region, by using a proof reading polymerase for amplification, and by implementing molecular mass modifiers for prevention of molecular mass overlaps. In a series of experiments, the performance of the multiplexed PCR-ICEMS assay was evaluated. The ICEMS-based DNA profiling assay was found to be competitive regarding detection sensitivity and analyzability of degraded and casework samples with commercially available electrophoretic typing approaches, which suggests that multiplexed PCR-ICEMS assays could represent a valuable tool for (forensic) genetics.

Effect of a carboxyl group on the chemiluminescent reaction of tris(2,2′-bipyridine)ruthenium(III) with aliphatic amines

The effect of a carboxyl group beside nitrogen of aliphatic amines on the tris(2,2′-bipyridine)ruthenium(III), Ru(bpy) 3 3+, chemiluminescent reaction was examined. It has been shown that a carboxylate anion promotes the chemiluminescent reaction at a lower pH and then the aliphatic amines with this substituent can be sensitively detected compared with corresponding aliphatic amines without this substituent. Based on this finding, preliminary studies on simultaneous determination of 4-hydroxyproline, N-methylglycine, N-methylalanine, proline, and pipecolic acid in human serum have been performed using isocratic reversed-phase ion-pair high-performance liquid chromatography (HPLC) with electrogenerated Ru(bpy) 3 3+ chemiluminescent detection. The detection limits (signal-to-noise ratio of 3) with the proposed method were 3.0, 12, 2.7, 4.6, and 10 nM for 4-hydroxyproline, N-methylglycine, N-methylalanine, proline, and pipecolic acid, respectively.

Comparison of Biotransformation of Inorganic Selenium by Lactobacillus and Saccharomyces in Lactic Fermentation Process of Yogurt and Kefir

The aim of the present study was to characterize, quantify, and compare the different selenium species that are produced when lactic fermentation with two different types of microorganisms, bacteria (Lactobacillus) and yeast (Saccharomyces), take place to produce yogurt and kefir, respectively, and to study the transformation process of these species as a function of time. These two dairy products were chosen for the study because they are highly consumed in different cultures. Moreover, the microorganisms present in the fermentation processes are different. While the bacteria Lactobacillus is the one responsible for yogurt fermentation, a partnership between bacteria and the yeast Saccharomyces causes kefir fermentation. A comparative study has been carried out by fermenting Se(IV) enriched milk in the presence of both types of microorganisms, where the concentration range studied was from 0.5 to 20 microg g (-1). Enzymatic extraction enabled selenium speciation profiles, obtained by anionic exchange and ion-pairing reversed phase high performance liquid chromatography (IP-RP-HPLC) with inductively coupled plasma mass spectrometry (ICP-MS) detection. Scanning electron microscopy (SEM) applied to the enriched samples showed segregated Se (0), at added concentrations higher than 5 microg g (-1). The main Se species formed depended on the type of microorganism involved in the fermentation process, SeCys 2 and MeSeCys being the main species generated in yogurt and SeMet in kefir. The results obtained are different for both kinds of samples. Lactic fermentation for yogurt produced an increment in selenocystine (SeCys 2) and Se-methylselenocysteine (MeSeCys), while fermentation to produce kefir also incremented the selenomethionine (SeMet) concentration. The Se species are stable for at least 10 and 15 days for kefir and yogurt, respectively. After this period, selenocystine concentration decreased, and the concentration of Se-methylselenocysteine was found to significantly increase.

Simultaneous Tracking of Sulfur Species in the Oxidation of Thiourea by Hydrogen Peroxide

We employ reversed-phase ion-pair high-performance liquid chromatography to quantitatively characterize the oxidation kinetics of thiourea oxidation by hydrogen peroxide. The HPLC technique makes it possible to monitor the concentrations of a variety of sulfur-containing species with different oxidation states and to elucidate the relative phase relations among them. The experimental results are in good agreement with simulations from an 8-step reaction mechanism.

Increased forensic efficiency of DNA fingerprints through simultaneous resolution of length and nucleotide variability by high-performance mass spectrometry

Short tandem repeat (STR) typing is the most powerful method for determining the origin of a sample for a number of molecular disciplines such as medical genetics, population genetics, tumor analysis, transplantation medicine, or forensic crime scene analysis. STR alleles are routinely differentiated based upon their fragment size by electrophoresis under denaturing conditions, which does not take nucleotide variability into consideration. This simplification leads to loss of biological information as the nature of the individual sequence motifs that build an STR is not described. An alternative detection platform would be mass spectrometry, which captures the underlying sequence variation by comparing the molecular masses of DNA fragments. Here, we demonstrate that the combination of ion-pair reversed-phase high-performance liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry (ICEMS) is able to simultaneously detect length and nucleotide variability in STRs. Overall, 21 forensically relevant STRs that are also used in other scientific fields were screened in an Austrian population sample for the occurrence of nucleotide variability within or close to the repeat region. A total of 11 of the investigated loci (SE33, D2S1338, vWA, D21S11, D3S1358, D16S539, D8S1179, D7S820, D13S317, D5S818, and D2S441) brought additional allele (sequence) variants. Forensic efficiency, as determined by typical statistical parameters, was significantly increased by 20 to 30%. The beauty of ICEMS-STR-analysis is the fact that it represents one of the few technological advancements that allows direct comparison of newly generated data with existing data such as stored in DNA databases, which have a retarding effect on new developments.

Development and validation of an assay for the quantitative determination of cladribine nucleotides in MDCKII cells and culture medium using weak anion‐exchange liquid chromatography coupled with tandem mass spectrometry

The development and validation of an assay for the quantitative analysis of cladribine mono-, di- and triphosphate (2-chloro, 2'-deoxyadenosine 5'-mono-, di- and triphosphate or 2CdAMP, 2CdADP and 2CdATP) in culture medium (Optimem) and cell lysate is described. Cladribine mono- and diphosphate reference compounds were obtained by thermal degradation of cladribine triphosphate. The reference compounds were characterized using ion-pairing reversed-phase high-performance liquid chromatography with ultraviolet detection. The bioanalytical assay for 2CdAMP, 2CdADP and 2CdATP is based on weak anion-exchange liquid chromatography coupled with tandem mass spectrometry in the positive ion mode (WAXLC/MS/MS). A fused-silica electrospray capillary was used instead of a stainless steel electrospray capillary to minimize adsorption of analytes and thus decrease variation in the analyte signals. Dynamic ranges of 1.11-27.7, 0.550-55.0 and 1.31-52.3 nM for 2CdAMP, 2CdADP and 2CdATP, respectively, were validated in culture medium and cell lysate. Optimem samples required stabilization with 30% methanol to prevent conversion of 2CdATP into 2CdAMP and 2CdADP. All intra- and interday accuracies and precisions were within +/-20%. The stability of the compounds was assessed under various analytically relevant conditions. The method was successfully used to investigate cladribine nucleotide transport in vitro in Madin-Darby canine kidney II (MDCKII) cells.

Multiplex PCR/Liquid Chromatography Assay for Screening of Subtelomeric Rearrangements

In idiopathic or nonspecific mental retardation, the overall rate of cryptic subtelomeric rearrangements is estimated to be about 5%. Development of cost-effective screening for subtelomeric deletions would help clinical geneticists to make specific diagnoses in children with idiopathic mental retardation. Current screening modalities include fluorescence in situ hybridization (FISH) using subtelomeric probes and PCR-based quantitative analyses. Reductions in the cost and turnaround time will make the complete screening of subtelomeric rearrangements more widely used in clinical settings. Recently, a versatile method, called the multiplex PCR/liquid chromatography assay (MP/LC), was developed to assess copy numbers in this assay. Multiple genomic regions are amplified using unlabeled primers, then separated by ion-pair reversed-phase high-performance liquid chromatography. In the present study, we developed an MP/LC-based subtelomeric screening system that involves 21 multiple reactions and validated the protocol by analyzing 16 publicly available cell lines with known cytogenetic abnormalities involving at least one subtelomere per patient. To confirm the validity of the MP/LC method, we analyzed these cell lines concurrently with array-based comparative genomic hybridization (array-CGH), which gives higher resolution than the conventional G-banding technique. Among those 16 samples, the results from MP/LC and array-CGH agreed with each other perfectly. In 2 of the 16 samples, MP/LC correctly revealed subtelomeric duplications that were detected by array-CGH but were undetected by conventional cytogenetics, demonstrating the sensitivity of the MP/LC assay. This system is expected to be useful for making specific diagnoses and in genetic counseling for children with idiopathic mental retardation, a sizable fraction of whom have subtelomeric rearrangements.