Ion-pair Liquid Chromatography Research Articles

Determination of Aminoglycosides by Ion-Pair Liquid Chromatography with UV Detection: Application to Pharmaceutical Formulations and Human Serum Samples

Determination of Aminoglycosides by Ion-Pair Liquid Chromatography with UV Detection: Application to Pharmaceutical Formulations and Human Serum Samples

Quantification of all B vitamins in a single run using ion-pair modified liquid chromatography with UV detection

A rapid and simple approach based on ion-pair liquid chromatography for the estimation of all B vitamins in a single run of 35 min using UV detector is developed. The estimation is based on the separation of all B vitamins on the C18 column using gradient elution comprising mobile phase A (65% methanol) and B (sodium dihydrogen phosphate, pH 3.05 containing 5 mM hexane sulphonic acid as an ion-pair reagent). Vitamins B1, B2, B3, B6, B9, and B12 were detected at 255 nm, while vitamins B5 and B7 were best detected at 200 and 210 nm, respectively. The standardized protocol was validated in buffer and milk permeate. The correlation coefficient for all B vitamins determined using a linear regression equation in the range of 1–30 µg mL−1 was > 0.99. The relative standard deviation for intra-day and inter-day repeatability of vitamins (10 µg mL−1) was less than 6%, and recoveries were in the 75–120% range. The applicability of the developed method has also been demonstrated for the quantification of B vitamins (B2, B3, B5, B6) in fortified commercial energy drinks.

Evaluation and correction of injection order effects in LC-MS/MS based targeted metabolomics

For large-scale and long-term metabolomics studies that involve a large batch or multiple batches of analyses, batch effects cause nonbiological systematic biases that may lead to false positive or false negative findings. Quantitative monitoring and correction of batch effects is critical to the development of reproducible and robust metabolomics platforms either for untargeted or targeted analyses. To achieve sufficient retention and separation of a broad range of metabolites with diverse chemical structures and physicochemical properties, LC-MS/MS based targeted metabolomics often involves 3 complemented chromatographic separation methods, including reversed-phase liquid chromatography (RP-LC), hydrophilic interaction liquid chromatography (HILIC), and ion-pair liquid chromatography (IP-LC). The purpose of this study is to quantitatively evaluate intra-batch variations or injection order effects of the RP-LC, HILIC, and IP-LC methods for targeted metabolomics analyses, and develop strategies to minimize intra-batch variations and correct injection order effects for problematic metabolites. Both RP-LC and HILIC methods exhibit robust intra-batch reproducibility in 0.2 µM standard mix QC, with ∼96 % of the measured metabolites showing acceptable intra-batch variations (<20 %); whereas, the intra-batch reproducibility for some metabolites in cell matrix QC may be compromised due to stability issue, suboptimal chromatographic retention, and/or matrix effects causing ionization suppression and/or retention instability. The IP-LC method exhibits significant injection order effects, which could be effectively corrected by the developed exponential models of signal drift trends as a function of injection order for individual targeted metabolites.

Analysis of impurity profiling of arbekacin sulfate by ion-pair liquid chromatography coupled with pulsed electrochemical detection and online ion suppressor-ion trap-time off light mass spectrometry

Ion-pair liquid chromatography with pulsed electrochemical detection (LC-PED) was established for the analysis of impurities in arbekacin (ABK) sulfate. APursuit pentafluorophenylpropyl (PFP) column was used as stationary phase. This novel method showed greater separation and sensitivity ability. In a representative ABK sample, 24 impurity peaks were detected in LC-PED, where of only 9 were monitored by a post-column derivatization method prescribed by the Japanese Pharmacopoeia (JP). For identification of the chemical structures of the impurities detected by LC-PED, LC-Mass Spectrometry (MS) was used. Two challenges had to be overcome in this work. The first was the transfer of the MS incompatible mobile phase to an MS compatibleone while maintaining the elution order of the peaks in the chromatograms. Previously reported approaches such as two-dimensional (2D)LC were hardly applicable in this case due to the lack of ultraviolet (UV) absorbing chromophores in ABK and its impurities. The sodium hydroxide solution was replaced by aqueous ammonia to adjust the pH of the mobile phase used in LC-PED. The other challenge encountered was the ion suppression effect caused by trifluoroacetic acid (TFA) and pentafluoroproponic acid (PFPA) in the mobile phase. Some strategies such as “TFA-fixed” and its modifications were tried, but they were inconvenient and severe contamination of the MS was observed. A cationself-regenerating suppressor (CSRS), which was originally designed for increasing analyte conductivityof ammonia and amines analysis in ion chromatography (IC), was coupled between the LC and Ion Trap-Time of Flight (IT-TOF)-MS and almost all TFA and PFPA in the mobile phase were removed. The limit of detection (LOD) of ABK in this integrated system improved significantly to 20 ng/mL. The chemical structures of the 28 impurities were elucidated and 15 impurities were reported for the first time.

The critical role of mobile phase pH in the performance of oligonucleotide ion-pair liquid chromatography-mass spectrometry methods.

The critical role of mobile phase pH in the performance of oligonucleotide ion-pair liquid chromatography-mass spectrometry methods.

Rapid and reliable analysis of underivatized amino acids in urine using tandem mass spectrometry

Nowadays there is a great interest in the development of fast and reliable methods for the determination of amino acids (AAs) in biological samples due to their biological importance. In the present work, a method based on the use of a guard column (gC) prior to tandem mass spectrometry (gC-MS/MS) is proposed for the determination of proteinogenic AAs in urine. Heptafluorobutyric acid (HFBA) is used as ion pairing reagent. Comparison of the gC-MS/MS method versus tandem mass spectrometry (MS/MS) in stand-alone mode showed improved sensitivity and peak shape, and solved some problems related to interfering compounds, with a total analysis time of 2.8 min. All the proteinogenic AAs were adequately determined using the gC-MS/MS method, except glutamic acid (Glu).To confirm quantitative results obtained with gC-MS/MS for individual AAs, an ion-pair liquid chromatography tandem mass spectrometry method (LC-MS/MS) has also been developed. Both methods (gC-MS/MS and LC-MS/MS) were validated using synthetic urine. For the gC-MS/MS method, LODs and LOQs values were found to be between 0.004 and 0.425 mg/L and 0.01 and 1.40 mg/L, respectively. Aspartic acid (Asp) showed the highest LOD and LOQ values (3 mg/L and 9 mg/L, respectively). A one-point standard addition method and internal standard normalization were used for the quantification because matrix effects were observed. L-alanine-1-13C (Ala-13C) and L-leucine-1-13C (Leu-13C) were used as isotopically labeled internal standards.To demonstrate the applicability of the gC-MS/MS method in the reliable determination of AAs in real samples, urine from eighteen healthy volunteers were analyzed using both gC-MS/MS and LC-MS/MS methods. Similar quantitative results were obtained for individual AAs with both of them. In addition, possible differences in AAs concentrations related to sex were checked, but the results did not show significant differences for the evaluated compounds.

Rapid determination of multiple aminoglycoside antibiotics in veterinary formulations by ion-pair chromatography coupled with evaporative light scattering detection

A fast and simple method for simultaneous determination of eight aminoglycoside antibiotics using ion-pairing liquid chromatography (IPLC) coupled with evaporative light scattering detection (ELSD) has been developed and validated. Separation of amikacin, apramycin, gentamicin (as a sum of gentamicin C1, C2 and C1A), kanamycin A, neomycin B, paromomycin, streptomycin and tobramycin was achieved using C18 column with H2O and MeOH/acetone, both with addition of nonafluoropentanoic acid. Limits of detection ranged between 1.2 µg/mL and 2.8 µg/mL. The total analysis time was 9.5 min. The developed method showed the suitability for the routine quality control of veterinary drugs (drops, powders and paraffin-based suspensions).

Assessing the impact of nonspecific binding on oligonucleotide bioanalysis.

Aim: Accurate and reliable quantification of oligonucleotides can be difficult, which has led to an increased focus on bioanalytical methods for more robust analyses. Recent advances toward mitigating sample losses on liquid chromatography (LC) systems have produced recovery advantages for oligonucleotide separations. Results & methodology: LC instruments and columns constructed from MP35N metal alloy and stainless steel columns were compared against LC hardware modified with hybrid inorganic-organic silica surfaces. Designed to minimize metal-analyte adsorption, these surfaces demonstrated a 73% increase in 25-mer phosphorothioate oligonucleotide recovery using ion-pairing reversed-phase LC versus standard LC surfaces, most particularly upon initial use. Conclusion: Hybrid silica chromatographic surfaces improve the performance, detection limits and reproducibility of oligonucleotide bioanalytical assays.

Aminoglycosides analysis optimization using ion pairing liquid chromatography coupled to tandem mass spectrometry and application on wastewater samples

Aminoglycosides are mostly used as veterinary antibiotics. In France, their consumption accounts for about 10% of all prescribed animal medicine. Due to their high polarity nature (log Kow < -3), they require chromatographic separation by hydrophilic interaction liquid chromatography or ion-pairing chromatography. This study presents the development of an ion pairing liquid chromatography with alkanesulfonates coupled to tandem mass spectrometry for the analysis of 10 aminoglycosides (spectinomycin, streptomycin, dihydrostreptomycin, kanamycin, apramycin, gentamicin, neomycin and sisomicin) in wastewater samples. The novelty of this method lies in the addition of the ion paring salt directly and only into the sample vial and not in the mobile phase, lowering the amount of salt added and consequently reducing signal inhibition. The optimized method was validated and showed satisfactory resolution, performances suitable with the analysis of aminoglycosides in wastewater samples, with limits of quantifications less than 10 ng/mL for most of the compounds, low matrix effects, high accuracy (85%-115% recoveries) and reproducibility (2%-12%RSD). It was then applied successfully to raw and treated wastewater samples.

Analytical development of seamless procedures on cation-exchange chromatography and ion-pair chromatography with high-precision mass spectrometry for short-chain peptides

We report comprehensive analytical procedures for characterization of underivatized short-chain peptides by wet chemical treatment and ion-pair liquid chromatography with electrospray ionization–mass spectrometry (LC/ESI–MS). The chromatographic separation via a porous graphitic carbon stationary column eluted using an acetonitrile solvent gradient revealed a positive correlation between the retention time and the degree of hydrophobicity reflected in a distinct relationship between methylene (–CH2–) elongation of amino acids. We also validated the chromatographic separation utilizing an InertSustain PFP column and high-resolution mass spectrometry of the Orbitrap system, which supports the analytical accuracy for identification of monoisotopic mass. Using the quantitative analysis on a Corona charged aerosol detector, the recovery of the underivatized short-chain peptide fraction was >94.0 ± 4.9% (n = 10) after purification via cation-exchange chromatography. By eliminating the inorganic and organic matrix effect, we can avoid ion suppression and enhancement to demonstrate accurate analytical evaluations with robust mass spectroscopic responses. The developed method provides new insight into short-chain peptide chemistry that can be utilized for numerous interdisciplinary research applications.

Xanthotoxin affects depression-related behavior and neurotransmitters content in a sex-dependent manner in mice

The present study aimed to evaluate xanthotoxin's influence on male and female Swiss mice's depression-like behaviors and investigate the potential mechanism of this effect. Naturally derived furanocoumarin (the Apiaceae family), xanthotoxin, administered acutely (12.5 mg/kg), diminished the immobility level in the forced swim test only in males. The immobility level was lower in females than males, which may be associated with a higher serotonin level in the female prefrontal cortex. A dose-dependent increase of serotonin and noradrenaline was reported in the reverse-phase ion-pair liquid chromatography in the female prefrontal cortex but not in the hippocampus. We suggest that xanthotoxin may exert antidepressant properties and affect males and females differently. The increasing level of serotonin in the male and female prefrontal cortex may underlie this effect.

Dynamic Inventory of Intermediate Metabolites of Cyanobacteria in a Diurnal Cycle.

SummaryCyanobacteria are gaining importance both as hosts for photoautotrophic production of chemicals and as model systems for studies of diurnal lifestyle. The proteome and transcriptome of cyanobacteria have been closely examined under diurnal growth, whereas the downstream effects on the intermediary metabolism have not received sufficient attention. The present study focuses on identifying the cellular metabolites whose inventories undergo dramatic changes in a fast-growing cyanobacterium, Synechococcus elongatus PCC 11801. We identified and quantified 67 polar metabolites, whose inventory changes significantly during diurnal growth, with some metabolites changing by 100-fold. The Calvin-Benson-Bassham cycle intermediates peak at midday to support fast growth. The hitherto unexplored γ-glutamyl peptides act as reservoirs of amino acids. Interestingly, several storage molecules or their precursors accumulate during the dark phase, dispelling the notion that all biosynthetic activity takes place in the light phase. Our results will guide metabolic modeling and strain engineering of cyanobacteria.

Determination of Amphetamine and Methamphetamine in Human Hair by Gas Chromatography Mass Spectrometry

This article develops a combined solid phase extraction (SPE) and gas chromatography – mass spectrometry (GC-MS) procedure for determining amphetamine-type stimulants Amphetamine (AM) and Methamphetamine (MA) in human hair. Hair samples were incubated in methanol containing 1% hydrochloric acid in 18 hours and then subjected to SPE. The obtained extracts were evaporated to dryness, derivatized with heptafluorobutyric anhydride (HFBA) at 70 °C for 30 minutes prior to GC–MS analysis. Gas chromatography mass spectrometry was run on HP5-MS column (30 m × 0.25 mm × 0.25 µm) with detector MS 5975C. Experimentally, the proposed method proved sensitive, simple and time-saving, but quite accurate with a low limit of detection (LOD = 0.05ng/mg) and quantitation (LOQ = 0.15ng/mg).&#x0D; Keywords:&#x0D; SPE, GC – MS, hair samples, amphetamine, methamphetamine.&#x0D; References&#x0D; [1] Ming-Ren Fuh, Ti-Yu Wu and Tzuen-Yeuan Lin, Determination of amphetamine and methamphetamine in urine by solid phase extraction and ion-pair liquid chromatography–electrospray–tandem mass spectrometry Talanta, 68 (3) (2006), 987-991. https://doi.org/10.1016/j.talanta.2005.06.057[2] Naresh C. Jain, Thomas C. Sneath, and Robert D. Budd, Rapid Gas-Chomatographic Determination of Amphetamine and Methamphetamine in urine, Clinical Chemistry, 20 (11) (1974) 1460-1462. https://doi.org/10.1093/clinchem/20.11.1460.[3] Dong-liang Lin, Rea-Ming Yin, Ray H. Liu, Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of Amphetamine, Methamphetamine, 3,4-Methylenedioxy- amphetamine and 3,4-Methylenedioxymethamphetamine in Human Hair and Hair Sections, Journal of Food and Drug Analysis, 13(2) (2005) 193-200. https://doi.org/10.38212/2224-6614.2526[4] María Jesús Tabernero, Maria Linda Felli, Ana María Bermejo, Marcello Chiarotti, Determination of ketamine and amphetamines in hair by LC/MS/MS, Anal Bioanal Chem, 395(2009), 2547–2557. https://doi.org/10.1007/s00216-009-3163-4.[5] D.V. Doan, D.Q. Huy, N.D. Hue, T.M. Tri, Determination of methamphetamine in urine samples by gas chromatography mass spectrometry combined with solid phase extraction technique, Journal of Science and Technology 47 (6) (2009) 53-58 (in Vietnamese).[6] Rodger L. Foltz, Allison F. Fentiman, Ruth B. Foltz, GC/MS Assays for Abused Drugs in Body Fluids, National Institute on Drug Abuse, Maryland, 1980.[7] AOAC, Appendix F: Guidelines for Standard Method Performance Requirements, AOAC official methods of analysis, Maryland, 2016.[8] Eunyoung Han, Martin P. Paulus, Marc Wittmann, Heesun Chung, Joon myong Song, Hair analysis and self-report of methamphetamine use by methamphetamine dependent individuals, Journal of Chromatography B, 879 (2011) 541–547. https://doi.org/10.1016/j.jchromb.2011.01.002.&#x0D; &#x0D; &#x0D;

Simultaneous determination of oxo- and thio-arsenic species using HPLC-ICP-MS

A method for simultaneous analysis of common oxo-arsenic species and their thio-analogues by coupling ion-pair liquid chromatography to inductively coupled plasma mass spectrometry was developed and validated. The chromatographic separation was performed using a C8 stationary phase and a mobile phase containing 1.2 mmol/L tetramethylammonium hydroxide, 4 mmol/L malonic acid, 6.2 mmol/L sodium butane-1-sulfonate, and 0.05 % (v/v) methanol. Under these conditions, the 11 arsenic species (arsenite, arsenate, dimethylarsinic acid, monomethylarsonic acid, arsenobetaine, arsenocholine, tetramethylarsonium cation, trimethylarsine oxide, dimethyldithioarsinic acid, monomethylmonothioarsinic acid, and trimethyarsine sulfide) were separated. The detection limits for these species ranged from 1.4 to 4.0 ng/g As. The method was applied to the analysis of fish, crustacean, mussel, cephalopod and mushroom samples. Thio-arsenic species DMDTAV were found in mussel Mytilus chilensis and cuttlefish Sepis pharaonis samples in very low contents of 23 and 21 ng/g As, respectively. All seafood samples contained predominantly arsenobetaine (330–8570 ng/g As); other As species were present at various contents from 7 to 325 ng/g As.

Skin exposure: requirements to methods determining active ingredient of pesticides in washings

The article presents results of studies exemplified by diquat on analysis concerning influence of lower limit value of quantitative assessment in washing sample for safety coefficient in exposure and in absorbed dose, if acting substance is absent in workplace ambient air samples and in dermal washings of workers. To control diquat in dermal washings, there is a method based on ion-pair liquid chromatography with ultraviolet detection (working wavelength 310 nm). To concentrate sample, cartridges for solid-phase extraction, containing ion exchange sorbent (Oasis MCX 6cc/500 mg), are used. Lower limit of assessment in washing sample — 0,15 micrograms. Experimentally set washing completeness is within range of 80–92%, standard deviation of repetition is 7,0% at most. The method created was tested in nature studies determining dermal exposure in workers subjected to 5 various preparations based on diquat dibromide when used for surface spraying from tractor and from aircraft. For lower limit of detection in washing sample (0,15 micrograms/washing), calculated risk value of exposure varied within 0,26–0,36; risk of absorbed dose was low — 0,23 (the allowable one ≤1). Findings are that present measuring methods which provide lower limit of detection 1 and 5 micrograms in washing sample could result in unallowable risk establishment even with absence of the substance in all samples of workplace air and dermal washings. The calculation formula suggested enables to give theoretic basis for requirements to lower limit of detecting active substances in dermal washing samples for evaluating risk of pesticides use in agriculture.

SEPARATION AND ANALYSIS OF AMLODIPINE/BENAZEPRIL COMBINATION IN CAPSULES BY A NOVEL ION PAIR LIQUID CHROMATOGRAPHY

Objective: The objective of this study was to develop and validate a novel ion-pair liquid chromatography method, in order to separate and assay of amlodipine/benazepril combination in capsules. This method was a fast, practical and additional choice in quality control laboratories.Methods: The chromatographic conditions comprised of a classical C18-type stationary phase (250 × 4.6 mm, 5μ), with a mobile phase consisting of: 45% of 10-3 M of cetrimide and 55% acetonitrile. The flow rate was 1 ml/min; the detection wavelength was at 242 nm, under ambient temperature.Results: The method was validated for linearity with correlation coefficients very close to one, the accuracy with mean recovery values between 95.0-105.0%, precision with relative standard deviations of the calculated concentrations less than 5.0% and specificity in the presence of degradation products and excipients.Conclusion: The results presented in this paper showed that the developed method was fast and applicable, for the separation and determination of amlodipine/benazepril combination in capsules.

Retention of Fluorescent Amino Acid Derivatives in Ion-pairing Reversed-phase Liquid Chromatography.

Recent studies have shown that pillar array columns enable fast and quantitative analysis of amino acids. However, hydrophilic amino acids still cannot be retained on pillar array columns since they have limited retention ability. Ion-pairing liquid chromatography is a promising means of increasing analyte retention. In this study, the effects of ion-pairing reagents on the retention of eight hydrophilic amino acids (histidine, asparagine, glutamine, serine, arginine, aspartic acid, glycine, and glutamic acid) derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) under reversed-phase conditions on a conventional ODS column were studied. Among the ion-pairing reagents investigated, tetraheptylammonium bromide proved to be the most effective for increasing analyte retention. With a mobile phase consisting of 20 mM citrate buffer (pH 6.3)-acetonitrile (100:40, v/v) and 2 mM tetraheptylammonium bromide, the retention times of the eight NBD-amino acids-except NBD-arginine-were longer than 19.4 min, which was the retention time of NBD-valine when eluted without an ion-pairing reagent. As NBD-valine was well retained on pillar array columns, the chromatographic conditions may thus be applied in the analysis of hydrophilic amino acids using pillar array columns.

Simultaneous analysis of spectinomycin, halquinol, zilpaterol, and melamine in feedingstuffs by ion-pair liquid chromatography–tandem mass spectrometry

A method for the simultaneous analysis of veterinary drug residues (spectinomycin, halquinol, and zilpaterol) and contaminants (melamine) in feedingstuffs by liquid chromatography–tandem mass spectrometry was developed. Method performance for all analytes was evaluated by reversed-phase liquid chromatography, reversed-phase with altered chemical equilibrium, and hydrophilic interaction (HILIC) as chromatographic modes. Validation was in accordance to Commission Decision 657/2002/CE, by considering the best chromatographic approach. Ion-pair liquid chromatography with C18 as stationary phase led to the lowest random uncertainties, effective analyte separation and shorter time of analysis. Low precision deviations and good recovery rates were obtained and thus method reliability and sensitivity could be consolidated. Method applicability was evaluated by the analysis of samples of feedingstuffs, such as cattle, pig, and poultry feeds, feed ingredients of both animal and vegetable origins, and mineral feeds. Some samples showed quantifiable concentrations of halquinol and zilpaterol, reinforcing the importance of this new analytical control method.

Elevated urinary excretion of free pyridinoline in Friesian horses suggests a breed-specific increase in collagen degradation

BackgroundFriesian horses are known for their high inbreeding rate resulting in several genetic diseases such as hydrocephaly and dwarfism. This last decade, several studies focused on two other presumed hereditary traits in Friesian horses: megaoesophagus and aortic rupture. The pathogenesis of these diseases remains obscure but an important role of collagen has been hypothesized. The purpose of this study was to examine possible breed-related differences in collagen catabolism. Urinary specimens from Friesian (n = 17, median age 10 years old) and Warmblood horses (n = 17, median age 10 years old) were assessed for mature collagen cross-links, i.e. pyridinoline (PYD) (=hydroxylysylpyridinoline/HP) and deoxypyridinoline (DPD) (lysylpyridinoline /LP).Solid-phase extraction was performed, followed by reversed-phase ion-paired liquid chromatography prior to tandem mass spectrometry (MS/MS) detection.ResultsMean urinary concentrations of free PYD, expressed as fPYD/creatinine ratio, were significantly higher in Friesian horses compared to Warmblood horses (28.5 ± 5.2 versus 22.2 ± 9.6 nmol/mmol, p = 0.02) while mean fDPD/creatinine ratios were similar in both horse breeds (3.0 ± 0.7 versus 4.6 ± 3.7 nmol/mmol, p = 0.09).ConclusionsSince DPD is considered a specific bone degradation marker and PYD is more widely distributed in connective tissues, the significant elevation in the mean PYD/DPD ratio in Friesian versus Warmblood horses (9.6 ± 1.6 versus 5.7 ± 1.8, p < 0.0001) suggests a soft tissue origin for the increased fPYD levels. Considering that a previous study found no differences in total collagen content between Friesian and Warmblood horses for tendon and aortic tissue, this indicates a higher rate of collagen degradation. The latter might, at least in part, explain the predisposition of Friesians to connective tissue disorders.

Determination of Antimicrobial Residues in Honey by Liquid Chromatography Tandem Mass Spectrometry

Antibiotics are generally used worldwide against bacterial diseases in the treatment of food-producing animals. Since the residues of active agents or their metabolites can appear in these foods, the European Union, for instance, has set maximum residue limit concentrations for authorised veterinary drugs in foodstuffs. However, as yet, regulatory limits have not been established for honey and thus far, only recommendations exist. The aim of this study is to present a multiscreening method for residues in honey for the determination of 36 antimicrobial residues associated with several antibiotics of the B1 group (sulfonamides, trimethoprim, aminoglycosides, tetracyclines, quinolones and lincomycin) as well as the antibiotic griseofulvin. During the screening analysis, samples are hydrolysed in an acidified medium, purified on polymeric solid-phase extraction cartridges and subsequently analysed by reversed phase ion pair liquid chromatography tandem mass spectrometry. The liquid chromatographic separation was optimised by computer simulation with DryLab software. The positive identification of target compounds in suspicious samples was confirmed using earlier developed antibiotic class specific methods of which the aminoglycoside method is herein described in detail. The developed approaches were then applied to samples in the national monitoring program after their successful validation. Moreover, the screening and confirmatory methods were applied to proficiency test samples resulting in satisfactory identification and quantification. However, the analysis of real samples revealed that co-eluting target compounds can have considerable influence on the accuracy of this semi-quantitative multiscreening method.