Creatinine in urine - a method comparison.

Drug screening in urine is widely applied in forensic toxicology. Contrary to blood analysis, excessive or reduced fluid intake can substantially alter the concentration of substances in urine. As a standard to detect urinary dilution, creatinine concentrations are analyzed. A sample with a concentration below 20 mg/dL is generally defined as too diluted to provide a valid result in abstinence control samples. This work investigates the potential of three different methods for the determination of creatinine concentrations in urine samples: A ZIC-HILIC based LC-MS/MS method, a spectrophotometric method on an AU 480 chemistry system, and a portable, immunoassay based point-of-care testing device was compared by measuring 200 urine samples. When comparing the two laboratory methods, LC-MS/MS and spectrophotometry, a mean difference of 3.7 ± 14 mg/dL was found, indicating that the spectrophotometric method is slightly overestimating the creatinine concentration. When comparing the LC-MS/MS method with the point-of-care testing device, a mean concentration difference within the calibration range for POCT (>20 mg/dL (excluding 16 samples) and <500 mg/dL (excluding 4 samples)) of 21 ± 37 mg/dL was found, indicating that the point-of-care testing device overestimates the measured creatinine concentration. A point-of-care testing device as used during this study can provide valuable information for on-site analysis. However, reported concentrations above 500 mg/dL should be further evaluated e.g. by dilution of the sample

Multiple parasite infections and their relationship to self-reported morbidity in a community of rural Côte d'Ivoire

Background Concomitant parasitic infections are common in the developing world, yet most studies focus on a single parasite in a narrow age group. We investigated the extent of polyparasitism and parasite associations, and related these findings to self-reported morbidity. Methods Inhabitants of 75 randomly selected households from a single village in western Côte d'Ivoire provided multiple faecal specimens and a single finger prick blood sample. The Kato-Katz technique and a formol-ether concentration method were employed to screen faecal samples for Schistosoma mansoni, soil-transmitted helminths and intestinal protozoa. Giemsa-stained blood smears were analysed for malaria parasites. A questionnaire was administered for collection of demographic information and self-reported morbidity indicators. Results Complete parasitological data were obtained for 500/561 (89.1%) participants, similarly distributed among sex, with an age range from 5 days to 91 years. The prevalences of Plasmodium falciparum, hookworms, Entamoeba histolytica/E. dispar, and S. mansoni were 76.4%, 45.0%, 42.2%, and 39.8%, respectively. Three-quarters of the population harboured three or more parasites concurrently. Multivariate analysis revealed significant associations between several pairs of parasites. Some parasitic infections and the total number of parasites were significantly associated with self-reported morbidity indicators. Conclusions Our data confirm that polyparasitism is very common in rural Côte d'Ivoire and that people have clear perceptions about the morbidity caused by some of these parasitic infections. Our findings can be used for the design and implementation of sound intervention strategies to mitigate morbidity and co-morbidit

Optimization of Sample Preparation for the Identification and Quantification of Saxitoxin in Proficiency Test Mussel Sample using Liquid Chromatography-Tandem Mass Spectrometry

Saxitoxin (STX) and some selected paralytic shellfish poisoning (PSP) analogues in mussel samples were identified and quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample extraction and purification methods of mussel sample were optimized for LC-MS/MS analysis. The developed method was applied to the analysis of the homogenized mussel samples in the proficiency test (PT) within the EQuATox project (Establishment of Quality Assurance for the Detection of Biological Toxins of Potential Bioterrorism Risk). Ten laboratories from eight countries participated in the STX PT. Identification of PSP toxins in naturally contaminated mussel samples was performed by comparison of product ion spectra and retention times with those of reference standards. The quantitative results were obtained with LC-MS/MS by spiking reference standards in toxic mussel extracts. The results were within the z-score of +/- 1 when compared to the results measured with the official AOAC (Association of Official Analytical Chemists) method 2005.06, pre-column oxidation high-performance liquid chromatography with fluorescence detection (HPLC-FLD).Peer reviewe

Online coupling of pure O2 thermo-optical methods – 14C AMS for source apportionment of carbonaceous aerosols

This paper reports on novel separation methods developed for the direct determination of 14C in organic carbon (OC) and elemental carbon (EC), two sub-fractions of total carbon (TC) of atmospheric air particulate matter. Until recently, separation of OC and EC has been performed off-line by manual and time-consuming techniques that relied on the collection of massive CO2 fractions. We present here two on-line hyphenated techniques between a Sunset OC/EC analyzer and a MICADAS (MIni radioCArbon DAting System) accelerator mass spectrometer (AMS) equipped with a gas ion source. The first implementation facilitates the direct measurement in the low sample size range (<10 lg C) with high throughput on a routine basis, while the second explores the potential for a continuous-flow real-time CO2 gas feed into the ion source. The performance achieved with reference materials and real atmospheric samples will be discussed to draw conclusions on the improvement offered in the field of 14C aerosol source apportionment

Results of a Saxitoxin Proficiency Test Including Characterization of Reference Material and Stability Studies

A saxitoxin (STX) proficiency test (PT) was organized as part of the Establishment of Quality Assurance for the Detection of Biological Toxins of Potential Bioterrorism Risk (EQuATox) project. The aim of this PT was to provide an evaluation of existing methods and the European laboratories' capabilities for the analysis of STX and some of its analogues in real samples. Homogenized mussel material and algal cell materials containing paralytic shellfish poisoning (PSP) toxins were produced as reference sample matrices. The reference material was characterized using various analytical methods. Acidified algal extract samples at two concentration levels were prepared from a bulk culture of PSP toxins producing dinoflagellate Alexandrium ostenfeldii. The homogeneity and stability of the prepared PT samples were studied and found to be fit-for-purpose. Thereafter, eight STX PT samples were sent to ten participating laboratories from eight countries. The PT offered the participating laboratories the possibility to assess their performance regarding the qualitative and quantitative detection of PSP toxins. Various techniques such as official Association of Official Analytical Chemists (AOAC) methods, immunoassays, and liquid chromatography-mass spectrometry were used for sample analyses.Peer reviewe

Automated High-Throughput Analysis of Tramadol and O-Desmethyltramadol in Dried Blood Spots.

The World Anti-Doping Agency (WADA) and the International Testing Agency (ITA) recently announced the development and implementation of dried blood spot (DBS) testing for routine analysis in time for the 2022 Winter Olympic and Paralympic Games in Beijing. Following the introduction of a ban on the use of Tramadol in competition in March 2019, the Union Cycliste International (UCI) started a pilot study for the manual analysis of Tramadol in DBS for anti-doping purposes. In this context, we present a fully automated LC-MS/MS-based method with automated sample preparation using a CAMAG DBS-MS 500 for the analysis of tramadol and its metabolite O-desmethyltramadol in DBS. The presented approach reduces manual handling in the laboratory to an absolute minimum, only requiring the preparation of calibration and quality control DBS cards. The method was developed, optimized, and validated before performing cross-validation with a liquid blood-based analysis method using authentic samples from forensic cases. During the validation process, the method showed an extraction efficiency of 62%, linearity r2 >0.99, accuracy and precision (within ±15% and ±20% at the LLOQ) for the determination of tramadol and O-desmethyltramadol. Method comparison in liquid blood with 26 samples showed good agreement (90±19% for tramadol and 94±14% for O-desmethyltramadol). In conclusion, automated analysis of tramadol and O-desmethyltramadol in DBS provides a fast and accurate solution for anti-doping screening. It is suited for high-throughput analysis, having a run time of about 4 min per sample. Furthermore, with the automated approach, manual sample extraction becomes obsolete

N-Acetyltaurine as a novel urinary ethanol marker in a drinking study

The forensic utility of N-acetyltaurine (NAcT) in urine as a marker for ethanol intake was examined. A HILIC-based liquid chromatography method for the mass spectrometric determination of NAcT, taurine, and creatinine in urine was developed and validated to investigate NAcT formation and elimination in a drinking study. Thereby, eight subjects ingested 0.66 to 0.84 g/kg alcohol to reach a blood alcohol concentration (BAC) of 0.8 g/kg. Blood and urine were taken every 1.5-2 h, during the first 8 h. NAcT and taurine levels were measured and corrected for the urine's dilution by normalization to a creatinine concentration of 1 mg/mL. For the determination of NAcT and taurine, uncorrected lower limits of quantitation (LLOQs) were at 0.05 μg/mL of urine. In the drinking study, NAcT proved to be an endogenous compound, which is present at a range of 1.0 to 2.3 μg/mL in urine of alcohol-abstinent subjects. Maximum NAcT concentrations were reached in samples taken 3 to 6 h after the start of drinking, whereby an upregulation in N-acetyltaurine could be found for all the subjects. The mean peak concentrations (c̅ max) of 14 ± 2.6 μg/mL (range 9-17.5 μg/mL) were reached. Within 24 h, the NAcT levels declined to endogenous concentrations. The detectability of NAcT was found to be slightly shifted compared to BAC: When BAC was not detectable anymore, NAcT levels were still elevated. After 24 h, when ethyl glucuronide (EtG) and ethyl sulfate (EtS) were still detectable, NAcT concentrations showed endogenous levels again. Positive NAcT results can be used as an indicator for recent alcohol consumption. A direct relationship between NAcT and taurine concentrations could not be found. Graphical abstract N-acetyltaurine concentrations for eight subjects during the first 24 h after an alcohol consumption of 0.8 g/kg

Fully Automated Determination of Phosphatidylethanol 16:0/18:1 and 16:0/18:2 in Dried Blood Spots.

PURPOSE Direct alcohol markers are widely applied during abstinence monitoring, driving aptitude assessments and workplace drug testing. The most promising direct alcohol marker was found to be phosphatidylethanol (PEth). Compared to other markers it shows a long window of detection due to accumulation in blood. To facilitate and accelerate the determination of PEth in DBS, we developed a fully automated analysis approach. METHODS The validated and novel online-SPE-LC-MS/MS method with automated sample preparation using a CAMAG DBS-MS 500 system reduces manual sample preparation to an absolute minimum, only requiring calibration and quality control DBS. RESULTS During the validation process, the method showed a high extraction efficiency (>88%), linearity (correlation coefficient >0.9953), accuracy and precision (within ±15%) for the determination of PEth 16:0/18:1 and PEth 16:0/18:2. Within a run time of about 7 min, the two monitored analogs could be baseline separated. A method comparison in liquid whole blood of 28 authentic samples from alcohol use disorder patients showed a mean deviation of less than 2% and a correlation coefficient of >0.9759. The comparison with manual DBS extraction showed a mean deviation of less than 8% and a correlation coefficient of >0.9666. CONCLUSIONS The automated analysis of PEth in DBS can provide a fast and accurate solution for abstinence monitoring. In contrast to the manual extraction of PEth in DBS, no laborious sample preparation is required with this automated approach. Furthermore, the application of the internal standard by a spray module can compensate for extraction bias and matrix effects

Fully Automated Correction for the Hematocrit Bias of Non-Volumetric Dried Blood Spot Phosphatidylethanol Analysis

The quantitative analysis of substances in dried blood spots (DBS) has gained vast popularity in the past decade. The World Anti-Doping Agency (WADA) also recently committed to implementing DBS. Currently, DBS sampling mainly has focused on various volumetric sampling devices such as Hemaxis, Capitainer, and Mitra. These devices are designed to collect a specific sample volume, independent of the hematocrit (HCT), to enable quantitative DBS analysis. Here, we present an automated solution that makes the necessity of volumetric sampling for quantitative DBS analysis obsolete. Combining automated reflectance-based HCT correction in combination with fully automated DBS LC-MS/MS analysis, the novel strategy permits high-throughput analysis in combination with HCT independence. Studying the model compound phosphatidylethanol 16:0/18:1, which is HCT-dependent due to incorporation into red blood cells, an implementation of DBS HCT normalization is presented. First, the performance of the automated HCT module with DBS is demonstrated compared to standardized HCT analysis from whole blood using a centrifuge. Second, the HCT dependency of fully automated PEth analysis from DBS is evaluated. Third, a solution to correct for the HCT dependency of PEth using the HCT scanner is presented. The study demonstrates that as soon as the HCT dependence of an analyte is known, a correction factor can be applied for the normalization of HCT levels. In the context of PEth, a linear increase in PEth concentration was observed, as the analyte is primarily located within the cellular fraction. Based on the obtained results, the use of a common correction factor for PEth DBS is possible