Min Run Time Research Articles

Development and Validation of a Novel Lateral Flow Immunoassay (LFIA) for the Rapid Screening of Paralytic Shellfish Toxins (PSTs) from Shellfish Extracts.

A single-step lateral flow immunoassay (LFIA) was developed and validated for the rapid screening of paralytic shellfish toxins (PSTs) from a variety of shellfish species, at concentrations relevant to regulatory limits of 800 μg STX-diHCl equivalents/kg shellfish meat. A simple aqueous extraction protocol was performed within several minutes from sample homogenate. The qualitative result was generated after a 5 min run time using a portable reader which removed subjectivity from data interpretation. The test was designed to generate noncompliant results with samples containing approximately 800 μg of STX-diHCl/kg. The cross-reactivities in relation to STX, expressed as mean ± SD, were as follows: NEO: 128.9% ± 29%; GTX1&4: 5.7% ± 1.5%; GTX2&3: 23.4% ± 10.4%; dcSTX: 55.6% ± 10.9%; dcNEO: 28.0% ± 8.9%; dcGTX2&3: 8.3% ± 2.7%; C1&C2: 3.1% ± 1.2%; GTX5: 23.3% ± 14.4% (n = 5 LFIA lots). There were no indications of matrix effects from the different samples evaluated (mussels, scallops, oysters, clams, cockles) nor interference from other shellfish toxins (domoic acid, okadaic acid group). Naturally contaminated sample evaluations showed no false negative results were generated from a variety of different samples and profiles (n = 23), in comparison to reference methods (MBA method 959.08, LC-FD method 2005.06). External laboratory evaluations of naturally contaminated samples (n = 39) indicated good correlation with reference methods (MBA, LC-FD). This is the first LFIA which has been shown, through rigorous validation, to have the ability to detect most major PSTs in a reliable manner and will be a huge benefit to both industry and regulators, who need to perform rapid and reliable testing to ensure shellfish are safe to eat.

A Quick Assay for the Quantitation of Deoxynivalenol in Grain Samples by Liquid Chromatography with UV Detection.

A quick and effective extraction of deoxynivalenol (DON) in food samples is described. The samples were extracted using 80% acetonitrile-water solution, followed by a second extraction using an 80% methanol-water solution. The extract was then subjected to an immunoaffinity column (IAC) cleanup. Quantification of isolated analyte was obtained by LC with UV detection at 220 nm. LC-UV parameters were optimized with a C18 LC column resulting in a 6 min run time. Certified reference materials (CRMs) of three different matrixes at three different levels were analyzed. Wheat, barley, and corn samples containing 0.5, 1.0, and 1.9 mg/kg, respectively, were analyzed. Recoveries from all CRM samples were calculated using an external standard curve (Figure 1) and ranged from 80 to 102%. Confirmation of DON in the sample extracts was accomplished using LC/MS. This method provides a rapid, specific, and robust assay for the analysis of DON with an uncertainty measurement of 12.2% and method detention limit of less than 0.125 mg/kg. Other methods that do not use an IAC produce dirty extracts that sometimes require washing the chromatographic column after each sample injection. The method presented here does not require washing the column, which leads to faster LC analysis time/sample.

Determination of thiocyanate in saliva by headspace gas chromatography-mass spectrometry, following a single-step aqueous derivatization with triethyloxonium tetrafluoroborate

A novel method for the determination of salivary thiocyanate is presented. Thiocyanate was converted into ethyl thiocyanate by single-step aqueous derivatization based on triethyloxonium tetrafluoroborate and measured by gas chromatography-mass spectrometry (15min runtime). The ethyl thiocyanate derivative is volatile and can be sampled from the headspace. The derivatization chemistry proposed allows for separation of the analyte from saliva matrix whose introduction in the measurement system is avoided. Quantitation of the analyte was obtained by isotope dilution, employing a 13C-enriched thiocyanate as internal standard. Technical details and fundamental aspects of derivatization chemistry and calibration strategy are presented.The method was validated by comparison with a standard method based on ion chromatography. The two independent methodologies produced results in agreement within 3%. Also a three level spike recovery test was carried out for validation purpose and quantitative recoveries were attained. The method is fast, simple, safe, and sensitive. Measurement of a 1mL volume 50ng/g of thiocyanate standard produced a signal-to-noise ratio of 250 for the analytical peak. This method is therefore suitable for ultra-trace determination of thiocyanate (low part-per-billion range). For the application described the full detection potential of the method was not required and the sample preparation presented has been designed for quantitation of saliva samples containing 1–400μg/g of thiocyanate with a combined standard uncertainty of 2% relative for saliva samples containing 25μg/g of thiocyanate. This method was applied for the determination of thiocyanate in human saliva samples.

Isolation and quantification of oligomeric and polymeric procyanidins in leaves and flowers of Hawthorn (Crataegus spp.)

Proanthocyanidins (PAs) constitute a class of polyphenols with flavan-3-ols as monomeric building blocks. These polyphenols are mostly quantified by colorimetric methods or by chromatographic determination of monomeric flavan-3-ols or low molecular oligomers as lead compounds. No reliable analytical methods are available for unambiguous identification of the homologues series of oligo- and polymeric PAs.For Hawthorn leaf and flower (Crataegi folium cum flore) from Crataegus spp. (Rosaceae) a protocol for preparative isolation of oligomeric and polymeric PAs from an acetone–water extract was developed, yielding procyanidin reference clusters with defined degree of polymerization (DP) from 2 to 10 besides a procyanidin-polymer. Identity and purity of these clusters were proven by HPLC, MS and in part NMR studies.For identification and quantification from Hawthorn an ICH-Q2 validated UHPLC method with fluorimetric detection and less than 10min runtime was developed. The method enabled quantification of procyanidin clusters with DP from 2 to 10 besides the polymer fraction. Batch analysis revealed procyanidin contents of about 20 to 45mg/g from a homologues series of oligomeric PAs and about 50% of polymer fraction.Monitoring of procyanidin distribution during seasonal growth of fresh plants of Crataegus monogyna showed more or less constant contents between 20 and 55mg/g dry weight of oligomeric procyanidins during the growing season in the different plant organs with strong accumulation in the flowers and fruits (55mg/g dry weight). From these data it can be speculated that procyanidins serve as part of the plants defense system in the reproductive organs of the plant.

Quantification of steroidal alkaloids in Buxus papillosa using electrospray ionization liquid chromatography–triple quadrupole mass spectrometry

Buxus papillosa is one of the most extensively studied species of the genus Buxus known to possess steroidal alkaloids which can be used for assessing the various pharmacological activities of this plant. This paper describes the liquid chromatography–electrospray ionization triple quadrupole mass spectrometry (LC–ESI-QQQ-MS) method for the quantification of six steroidal alkaloids as chemical markers in the extracts of leaves, roots and stems of B. papillosa. Quantitative MS/MS analysis was carried out by optimization of the most sensitive transition for each analyte. This has yielded detection and quantification limits of 0.486–8.08ng/mL and 1.473–24.268ng/mL, respectively for all analytes. Linearity of response was also achieved and the regression coefficient found to be >0.99 for all analyzed compounds. The newly developed MRM (Multiple Reaction Monitoring) method showed excellent sensitivity for the quantification of steroidal alkaloids within 15min run time. This paper describes the application of LC–QQQ-MS technique for steroidal alkaloids analysis in plant samples.

A Rapid, Robust and Ultra-Sensitive HPLC Enantioseparation of β-Amino Alcohols.

Enantioseparation of amino acid analogues racemic β-amino alcohols has hitherto been thought impossible without prior derivatization. Method developers of chromatographic enantioseparation often face detection challenge due to low ultraviolet (UV) absorbance of these molecules. A new chiral derivatizing reagent, benzimidazole-(S)-naproxen amide, was synthesized to provide UV detectable chiral moiety for detection of respective diastereomers of eight β-amino alcohols (dl-alaninol, dl-leucinol, dl-prolinol, dl-phenylalaninol, dl-phenylglycinol, dl-valinol, dl-homophenylalaninol and dl-methioninol). An HPLC method has been developed and validated and both the diastereomers were separated within 31 min runtime. Excellent resolution to a pmol detection limit level was achieved. The developed HPLC method for enantioseparation of β-amino alcohols is the first-ever report of its own kind having such a high detection sensitivity.

A fast liquid chromatography-mass Spectrometry methodology for membrane lipid profiling through hydrophilic interaction liquid chromatography

In this paper, we report the development of a new method based on HILIC-ESI-MS for the separation of several different membrane lipid classes and their detection on a triple quadrupole mass spectrometer using Precursor Ion (PIS) and Neutral Loss (NL) scanning in positive ion mode. Four different columns were tested for their ability to separate, under different conditions, a mixture of 14 lipid standards containing 7 glycerophospholipids (GPL), 2 glycosphyngolipids (GSL), 3 glycolipids (GL) and 2 betaine lipids (BL).The best separation was obtained using a Lichrosphere DIOL column as stationary phase and water (10mm ammonium acetate)/acetonitrile gradient elution as mobile phase which allows the separation of the 14 lipid classes within 35min runtime. Our method was successfully tested for the separation and analysis of crude lipid extracts obtained from a green alga (Jaoa bullata), a dinoflagellate (Peridinium cinctum) and a plant (Vitis vinifera cv. Corvina)

Lignin yield maximization of mixed biorefinery feedstocks by organosolv fractionation using Taguchi Robust Product Design

Lignin, isolated from switchgrass (Panicum virgatum) and tulip poplar (Liriodendron tulipifera) using organosolv fractionation is currently being explored for its potential use in the production of value-added chemicals and bio-based polymers. Taguchi Robust Product Design (TRPD) was applied to maximize lignin yield from the fractionation process. The following four controllable design factors were used in the TRPD: process temperature (120 °C, 140 °C and 160 °C), fractionation time (56 and 90 min), sulfuric acid concentration (0.025 M, 0.05 M and 0.1 M), and feedstock type (switchgrass/tulip poplar chip ratios of 10/90, 50/50 and 90/10). Process noise was induced in the experiment by using either the mass- or volume-based feedstock charges of switchgrass and tulip poplar chips. A maximum mean lignin yield of 78.63 wt% and signal-to-noise ratio of 37.90 was found at a 90 min runtime, a process temperature of 160 °C, a sulfuric acid concentration of 0.1 M, and a feedstock composition of 10% switchgrass and 90% tulip poplar. Process temperature was the most significant factor that influenced lignin yield. This study may provide a pathway for industrialists and researchers interested in maximizing lignin yield in the organosolv fractionation process.

A Simple Bioanalytical Method for the Quantification of Levetiracetam in Human Plasma and Saliva

A novel, sensitive and selective ultra-high-performance liquid chromatography - coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method was developed and validated for the quantification of levetiracetam (LEV), a broad-spectrum antiepileptic drug, in human plasma and saliva. A simple protein precipitation method with acetonitrile as precipitating solvent was used to extract LEV from human plasma and saliva. LEV and an internal standard (fluconazole, IS) were separated on a Kinetex analytical column (5 μm C18 100A, 100 × 2.1 mm) using an isocratic solvent system consisting of methanol, water and 100% formic acid at a ratio of 97:3:0.25 (v/v/v) and a flow rate of 0.2 ml/min over 2 min run time. Detection was performed using target ions of [M+H]+ at m/z 171.0 for LEV and m/z 307.0 for IS in selective ion mode. The method was validated in the calibration range of 1.0-50.0 μg/mL for plasma and 0.5-30.0 μg/mL for saliva. Thus the present HPLC-MS/MS method for determination of LEV in human plasma and saliva, is highly sensitive, rapid with a short run-time of 2 min, can be suitable for high sample throughput. The developed method required minimal sample preparation and less plasma sample volume compared to earlier published LC-MS/MS methods. The validation parameters were found to be well within the acceptance limit.

Analysis of new designer drugs in post-mortem blood using high-resolution mass spectrometry.

An analytical method was developed and validated for the purpose of detecting and quantifying 37 new designer drugs including cathinones, hallucinogenic phenethylamines and piperazines. Using only 100 µL whole blood, a salting-out-assisted liquid-liquid extraction with acetonitrile was performed to isolate target compounds followed by chromatographic separation using a Waters ACQUITY ultra performance liquid chromatograph coupled to a Waters XEVO quadrupole time-of-flight mass spectrometer. Mephedrone-d3 was used as an internal standard. A gradient elution was used in combination with a Waters ACQUITY HSS C18 column (2.1 × 150 mm, 1.8 µm). Samples were analyzed using the detector in positive electrospray ionization mode with MS(E) acquisition. All compounds of interest were resolved in a 15 min run time and positively identified based on accurate mass of the molecular ion, two product ions and retention time. All analyte calibration curves were linear over the range of 0.05-2 mg/L with most correlation coefficient (r(2)) values >0.98. The limits of detection were within the range of 0.007-0.07 mg/L and limits of quantification within 0.05-0.1 mg/L. All analytes were stable 48 h after extraction and most were stable in blood after 1 week stored in a refrigerator and 3 freeze-thaw cycles. No carryover was observed up to 10 mg/L and no interferences from common therapeutic drugs or endogenous compounds. Recoveries ranged from 71 to 100% and matrix effects were assessed for blank, post-mortem and decomposed blood. All bias and % coefficient of variation values were within the acceptable values of ±15 and ≤15%, respectively (±20 and ≤20% at lower limit of quantification). The method was applied to several forensic cases where the subject exhibited behavior characteristic of designer drug intoxication and where routine screening for a panel of drugs was negative.

A simple and rapid method based on liquid chromatography-tandem mass spectrometry for the measurement of α-L-iduronidase activity in dried blood spots: an application to mucopolysaccharidosis I (Hurler) screening.

BackgroundWe developed an analytical method to measure α-L-iduronidase (IDUA) activity in dried blood spots. This was achieved by using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) with electrospray ionization in the positive ion mode.MethodsChromatographic separation was completed using mobile phase involving water-formic acid and acetonitrile-formic acid over 2.8 min of run time on a column with a Kinetex XB-C18 (Phenomenex, USA). The detection of column effluent was performed using a Xevo TQ-S triple quadrupole mass spectrometer (Waters, USA) in the multiple-reaction monitoring mode. This method was verified with blank and control samples at four activity levels: base, low, medium, and high. Control materials were provided from Centers for Disease Control and Prevention (CDC).ResultsIntra- and inter-day precisions were between 2.6% and 16.5% and between 7.9% and 17.0%, respectively. A correlative regression study on the IDUA activity in CDC-control samples performed to assess the validity of the developed method showed a highly significant linear association (r2=0.9976) between the calculated and CDC-reported values and an obvious difference in activity among the four levels. This reliable analytical method was applied to mucopolysaccharidosis I (Hurler) screening of patients under treatment (n=4) and in normal controls (n=129). IDUA activity ranged from 8.98 to 77.12 µmol/hr/L) in normal controls, and patients undergoing medical treatment showed low IDUA activity.ConclusionsThis method had advantages of simplicity, rapid sample preparation, and liquid chromatographic separation, which efficiently inhibited ionization suppression induced by matrix effects in mass spectrometric detection.

Determination of oxadixyl in wines by liquid chromatography- tandem mass spectrometry: single-laboratory and interlaboratory validation study.

A direct injection LC/MS/MS method for the determination of the pesticide oxadixyl in wines was developed and validated. A sample divert valve was used to deliver the fraction that contained oxadixyl to the mass spectrometer's electrospray ionization source. Oxadixyl was monitored and quantitated using two transitions in multiple reaction monitoring mode. The method demonstrated recoveries of 99.2 ± 2.0 and 96.7± 5.2% for red and white wines, respectively, a linearity range of 2-20 μg/L, LOD at 0.7 μg/L, LOQ of 2.0 μg/L, and precision values of 8.2% (RSDr) and 6.2% (RSDR). Direct injection of the wine onto a C18 ultra-performance LC column allowed automation and high throughput screening. Benefits of this approach includeminimal sample preparation, short (3 min) run times, and the use of matrix-matched calibration standards, which minimize the matrix effect due to interferences from wine phenolics, sugars, and various other components. The method's performance characteristics were not statistically different for white and red wines. An additional interlaboratory validation study involved 12 laboratories and demonstrated gooddata agreement with HorRat values ranging from 0.23 to 0.52.

Development and validation of a UPLC method for quantification of antiviral agent, Acyclovir in lipid-based formulations

PurposeThe objective of the current study is to evaluate the Ultra Performance Liquid Chromatography (UPLC) method for quantification of Acyclovir in lipid-based formulations. MethodA simple, rapid, reliable and precise reversed phase UPLC method has been developed and validated according to the regulatory guidelines, which composed of isocratic mobile phase; 0.25% formic acid (FA) in Milli-Q water with a flow rate of 0.5ml/min, and column BEH C18 (2.1×50mm, 1.7μm). The detection was carried out at 254nm. ResultsThe developed UPLC method was found to be rapid (1.2min run time), selective with well resoluted Acyclovir peak (0.89min) from different lipid matrices and sensitive (Limit of Detection (LOD) was 0.3ppm and Lower Limit of Quantification (LLOQ) was 1ppm). The accuracy and precision were determined and were perfectly matching with the standard FDA limits. ConclusionThe study showed that the proposed UPLC method can be used for the assessment of drug purity, stability, solubility and lipid-formulation release profile with no interference of excipients or related substances of active pharmaceutical ingredient.

Determination of hydroxytyrosol and tyrosol by liquid chromatography for the quality control of cosmetic products based on olive extracts

An analytical method for the simultaneous determination of hydroxytyrosol and tyrosol in different types of olive extract raw materials and cosmetic cream samples has been developed. The determination was performed by liquid chromatography with UV spectrophotometric detection. Different chromatographic parameters, such as mobile phase pH and composition, oven temperature and different sample preparation variables were studied. The best chromatographic separation was obtained under the following conditions: C18 column set at 35°C and isocratic elution of a mixture ethanol: 1% acetic acid solution at pH 5 (5:95, v/v) as mobile phase pumped at 1mLmin−1. The detection wavelength was set at 280nm and the total run time required for the chromatographic analysis was 10min, except for cosmetic cream samples where 20min runtime was required (including a cleaning step). The method was satisfactorily applied to 23 samples including solid, water-soluble and fat-soluble olive extracts and cosmetic cream samples containing hydroxytyrosol and tyrosol. Good recoveries (95–107%) and repeatability (1.1–3.6%) were obtained, besides of limits of detection values below the μgmL−1 level. These good analytical features, as well as its environmentally-friendly characteristics, make the presented method suitable to carry out both the control of the whole manufacture process of raw materials containing the target analytes and the quality control of the finished cosmetic products.

Liquid chromatography–tandem mass spectrometry method for the estimation of adefovir in human plasma: Application to a pharmacokinetic study

An analytical method based on solid phase extraction was developed and validated for analysis of adefovir in human plasma. Adefovir-d4 was used as an internal standard and Synergi MAX RP80A (150mm×4.6mm, 4µm) column provided the desired chromatographic separation of compounds followed by detection with mass spectrometry. The method used simple isocratic chromatographic condition and mass spectrometric detection in the positive ionization mode. The calibration curves were linear over the range of 0.50–42.47ng/mL with the lower limit of quantitation validated at 0.50ng/mL. Matrix effect was assessed by post-column infusion experiment to monitor phospholipids and post-extraction addition experiment was performed. The degree of matrix effect for adefovir was determined as 7.5% and ion-enhancement in five different lots of human plasma was 7.1% and had no impact on study samples analysis with 4.5min run time. The intra- and inter-day precision values were within 7.7% and 7.8%, respectively, for adefovir at the lower limit of quantification level. Validated bioanalytical method was successfully applied to clinical sample analysis.

Enhanced lipid isomer separation in human plasma using reversed-phase UPLC with ion-mobility/high-resolution MS detection

An ultraperformance LC (UPLC) method for the separation of different lipid molecular species and lipid isomers using a stationary phase incorporating charged surface hybrid (CSH) technology is described. The resulting enhanced separation possibilities of the method are demonstrated using standards and human plasma extracts. Lipids were extracted from human plasma samples with the Bligh and Dyer method. Separation of lipids was achieved on a 100 × 2.1 mm inner diameter CSH C18 column using gradient elution with aqueous-acetonitrile-isopropanol mobile phases containing 10 mM ammonium formate/0.1% formic acid buffers at a flow rate of 0.4 ml/min. A UPLC run time of 20 min was routinely used, and a shorter method with a 10 min run time is also described. The method shows extremely stable retention times when human plasma extracts and a variety of biofluids or tissues are analyzed [intra-assay relative standard deviation (RSD) <0.385% and <0.451% for 20 and 10 min gradients, respectively (n = 5); interassay RSD <0.673% and <0.763% for 20 and 10 min gradients, respectively (n = 30)]. The UPLC system was coupled to a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer, equipped with a traveling wave ion-mobility cell. Besides demonstrating the separation for different lipids using the chromatographic method, we demonstrate the use of the ion-mobility MS platform for the structural elucidation of lipids. The method can now be used to elucidate structures of a wide variety of lipids in biological samples of different matrices.

High-performance liquid chromatography determination of red wine tannin stickiness.

Red wine astringency is generally considered to be the sensory result of salivary protein precipitation following tannin-salivary protein interaction and/or tannin adhering to the oral mucosa. Astringency in red wine is often described using qualitative terms, such as hard and soft. Differences in qualitative description are thought to be due in part to the tannin structure. Tannin chemistry contributions to qualitative description have been shown to correlate with the enthalpy of interaction between tannin and a hydrophobic surface. On the basis of these findings, a method was developed that enabled the routine determination of the thermodynamics of the tannin interaction with a hydrophobic surface (polystyrene divinylbenzene) for tannins in red wine following direct injection. The optimized analytical method monitored elution at four different column temperatures (25-40 °C, in 5 °C increments), had a 20 min run time, and was monitored at 280 nm. The results of this study confirm that the calculated thermodynamics of the interaction are intensive and, therefore, provide specific thermodynamic information. Variation in the enthalpy of interaction between tannin and a hydrophobic surface (tannin stickiness) is a unique, concentration-independent analytical parameter. The method, in addition to providing information on tannin stickiness, provides the tannin concentration.

Optimised extraction of heterocyclic aromatic amines from blood using hollow fibre membrane liquid-phase microextraction and triple quadrupole mass spectrometry

Heterocyclic aromatic amines (HCA) are carcinogenic mutagens formed during cooking of proteinaceous foods, particularly meat. To assist in the ongoing search for biomarkers of HCA exposure in blood, a method is described for the extraction from human plasma of the most abundant HCAs: 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) (and its isomer 7,8-DiMeIQx), using hollow fibre membrane liquid-phase microextraction. This technique employs 2.5cm lengths of porous polypropylene fibres impregnated with organic solvent to facilitate simultaneous extraction from an alkaline aqueous sample into a low volume acidic acceptor phase. This low cost protocol is extensively optimised for fibre length, extraction time, sample pH and volume. Detection is by UPLC–MS/MS using positive mode electrospray ionisation with a 3.4min runtime, with optimum peak shape, sensitivity and baseline separation being achieved at pH 9.5. To our knowledge this is the first description of HCA chromatography under alkaline conditions. Application of fixed ion ratio tolerances for confirmation of analyte identity is discussed. Assay precision is between 4.5 and 8.8% while lower limits of detection between 2 and 5pg/mL are below the concentrations postulated for acid-labile HCA-protein adducts in blood.

Development and validation of an ultra high performance liquid chromatography tandem mass spectrometry method for simultaneous determination of sulfonamides, quinolones and benzimidazoles in bovine milk.

A simple, sensitive and reliable analytical method was developed for the simultaneous determination of 38 veterinary drugs (18 sulfonamides, 11 quinolones and 9 benzimidazoles) and 8 metabolites of benzimidazoles in bovine milk by ultra high performance liquid chromatography-positive electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS). Samples were extracted with acidified acetonitrile, cleaned up with Oasis(®) MCX cartridges, and analyzed by LC-MS/MS on an Acquity UPLC(®) BEH C18 column with gradient elution. The method allows such multi-analyte measurements within a 13min runtime while the specificity is ensured through the MRM acquisition mode. The method was validated according to the European Commission Decision 2002/657/EC determining specificity, decision limit (CCα), detection capability (CCβ), recovery, precision, linearity and stability. For compounds which have MRLs in bovine milk, the CCα values fall into a range from 11 to 115μg/kg, and the CCβ values fall within a range of 12-125μg/kg. For compounds which have not MRLs in bovine milk, the CCα values fall into a range from 0.01 to 0.08μg/kg, and the CCβ values fall within a range of 0.02-0.11μg/kg. The mean recoveries of the 46 analytes were between 87 and 119%. The calculated RSD values of repeatability and within-laboratory reproducibility experiments were below 11% and 15% for the 46 compounds, respectively. The method was demonstrated to be suitable for the simultaneous determination of sulfonamides, quinolones and benzimidazoles in bovine milk.

High-resolution droplet-based fractionation of nano-LC separations onto microarrays for MALDI-MS analysis.

We present a robust droplet-based device, which enables the fractionation of ultralow flow rate nanoflow liquid chromatography (nano-LC) eluate streams at high frequencies and high peak resolution. This is achieved by directly interfacing the separation column to a micro T-junction, where the eluate stream is compartmentalized into picoliter droplets. This immediate compartmentalization prevents peak dispersion during eluate transport and conserves the chromatographic performance. Subsequently, nanoliter eluate fractions are collected at a rate of one fraction per second on a high-density microarray to retain the separation with high temporal resolution. Chromatographic separations of up to 45 min runtime can thus be archived on a single microarray possessing 2700 sample spots. The performance of this device is demonstrated by fractionating the separation of a tryptic digest of a known protein mixture onto the microarray chip and subsequently analyzing the sample archive using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Resulting peak widths are found to be significantly reduced compared to standard continuous flow spotting technologies as well as in comparison to a conventional nano-LC-electrospray ionization-mass spectrometry interface. Moreover, we demonstrate the advantage of our high-definition nanofractionation device by applying two different MALDI matrices to all collected fractions in an alternating fashion. Since the information that is obtained from a MALDI-MS measurement depends on the choice of MALDI matrix, we can extract complementary information from neighboring spots containing almost identical composition but different matrices.