Comparison of a two-step Tempus600 hub solution single-tube vs. container-based, one-step pneumatic transport system.

OBJECTIVES: Efficient and timely transportation of clinical samples is pivotal to ensure accurate diagnoses and effective patient care. During the transportation process, preservation of sample integrity is crucial to avoid pre-analytical aberrations on laboratory results. Here, we present a comparative analysis between a two-step Tempus600 hub solution single-tube and a one-step, container-based pneumatic transport system (PTS) from Airco, for the in-house transportation of blood samples. METHODS: Ten blood samples from healthy volunteers were split in 10 mL collection tubes filled at full or half capacity for transportation with the two PTS (about 250 m). To compare the impact of transportation, markers of hemolysis such as lactate dehydrogenase (LDH), potassium (K+), and the hemolysis index (HI), were determined. Additionally, differences in HI in routine samples and repeated transportation was investigated. To assess and compare the mechanistic impact profiles, we recorded the acceleration profiles of the two PTS using a shock data logger. RESULTS: Transportation using the Tempus600 hub solution resulted in 49 and 46 % higher HI with samples filled to total or half capacity, respectively. Routine samples transported with the Tempus600 hub solution showed a higher median HI by 23 and 33 %. Additionally, shock logger analysis showed an elevated amount of shocks (6.5 fold) and shock intensities (1.8 fold). CONCLUSIONS: The Tempus600 hub solution caused an increased number of unreportable LDH or K+ results based on the hemolysis index. However, it was only statistically significant for LDH (p<0.01 and p<0.08) - while the comparisons for K+ were not statistically significant (p<0.28 and p<0.56).

In-depth evaluation of automated non-contact reflectance-based hematocrit prediction of dried blood spots.

Dried blood spot(s) (DBS) microsampling has increasingly attracted interest as a patient-centric alternative to conventional blood withdrawal. Despite the many advantages associated with DBS sampling, its widespread use in clinical practice is still hampered, which is mainly caused by the hematocrit (Hct) effect. One approach to cope with this issue is the Hct prediction of DBS using ultraviolet-visible (UV-Vis) spectroscopy. Recently, a UV-Vis-based Hct prediction module has been incorporated into the automated CAMAG® DBS-MS 500 HCT system. However, although a proof-of-principle yielded promising results, there is no formal in-depth evaluation of the performance of this module. Hence, it remained to be established to what extent automated Hct prediction of DBS via this module can universally be applied and generates acceptable results. Using authentic patient samples, we set up and validated a calibration model and evaluated whether this could serve as a 'generic' calibration model for different, independent Hct prediction modules. A quadratic calibration curve with 1/x2 weighting was established. The bias, intra-day and total precision were below 0.025 L L-1, 2.2% and 2.7%, respectively. Additionally, the influence of storage and the robustness of the method was evaluated. Moreover, a lab-lab comparison of the performance of the Hct module of two independently operated instruments demonstrated that the validated model can be used as a generic calibration model. Finally, application of the method to venous DBS (n = 48) prepared from patient samples in the context of therapeutic drug monitoring of tacrolimus revealed a good concordance between the actual (i.e. Sysmex-based) and UV-Vis-based predicted Hct.

Addressing New Possibilities and New Challenges: Automated Nondestructive Hematocrit Normalization for Dried Blood Spots.

ABSTRACT: The patient's hematocrit (HCT) level can adversely affect the analysis results when dried blood spots (DBS) are used for sampling. Volumetric DBS sampling has been proposed to nullify the impact of HCT area bias (spreading area) on DBS by normalizing to a known sample volume. However, this strategy ignores DBS-related parameters such as analyte properties (red blood cell-to-plasma ratio) and HCT recovery bias. With the recent release of fully automated HCT measurement systems for DBS analysis, a broad range of end users are now able to measure and correct a sample's HCT level in a nondestructive manner. These systems permit correction for all known HCT-related impacts on DBS, such as analyte properties, HCT recovery bias, HCT area bias, and venous blood-to-DBS ratio, supporting and accelerating future quantitative DBS applications. However, with these novel tools, new questions arise concerning the normalization of analytical results, the choice of technique (single-wavelength reflectance vs near-infrared spectroscopy), and the DBS card-handling process post sampling. Herein, the necessary considerations for end users are addressed and examples are provided.

Introduction of sample tubes with sodium azide as a preservative for ethyl glucuronide in urine.

Ethyl glucuronide (EtG) is a direct alcohol marker, which is widely used for clinical and forensic applications, mainly for abstinence control. However, the instability of EtG in urine against bacterial degradation or the post-collectional synthesis of EtG in contaminated samples may cause false interpretation of EtG results in urine samples. This study evaluates the potential of sodium azide in tubes used for urine collection to hinder degradation of ethyl glucuronide by bacterial metabolism taking place during growth of bacterial colonies. The tubes are part of a commercial oral fluid collection device. The sampling system was tested with different gram-positive and gram-negative bacterial species previously observed in urinary tract infections, such as Escherichia coli, Staphylococcus aureus, Enterecoccus faecalis, Staphylococcus epidermidis, Klebsiella pneumoniae, Enterobacter cloacae, and Pseudomonas aeruginosa. Inhibition of bacterial growth by sodium azide, resulting in lower numbers of colony forming units compared to control samples, was observed for all tested bacterial species. To test the prevention of EtG degradation by the predominant pathogen in urinary tract infection, sterile-filtered urine and deficient medium were spiked with EtG, and inoculated with E. coli prior to incubation for 4 days at 37 °C in tubes with and without sodium azide. Samples were collected every 24 hours, during four consecutive days, whereby the colony forming units (CFU) were counted on Columbia blood agar plates, and EtG was analyzed by LC-MS/MS. As expected, EtG degradation was observed when standard polypropylene tubes were used for the storage of contaminated samples. However, urine specimens collected in sodium azide tubes showed no or very limited bacterial growth and no EtG degradation. As a conclusion, sodium azide is useful to reduce bacterial growth of gram-negative and gram-positive bacteria. It inhibits the degradation of EtG by E. coli and can be used for the stabilization of EtG in urine samples.

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.

Determination of fatty acid ethyl esters in dried blood spots by LC-MS/MS as markers for ethanol intake: application in a drinking study.

The forensic utility of fatty acid ethyl esters (FAEEs) in dried blood spots (DBS) as short-term confirmatory markers for ethanol intake was examined. An LC-MS/MS method for the determination of FAEEs in DBS was developed and validated to investigate FAEE formation and elimination in a drinking study, whereby eight subjects ingested 0.66-0.84 g/kg alcohol to reach blood alcohol concentrations (BAC) of 0.8 g/kg. Blood was taken every 1.5-2 h, BAC was determined, and dried blood spots were prepared, with 50 µL of blood, for the determination of FAEEs. Lower limits of quantitation (LLOQ) were between 15 and 37 ng/mL for the four major FAEEs. Validation data are presented in detail. In the drinking study, ethyl palmitate and ethyl oleate proved to be the two most suitable markers for FAEE determination. Maximum FAEE concentrations were reached in samples taken 2 or 4 h after the start of drinking. The following mean peak concentrations (c̅(max)) were reached: ethyl myristate 14 ± 4 ng/mL, ethyl palmitate 144 ± 35 ng/mL, ethyl oleate 125 ± 55 ng/mL, ethyl stearate 71 ± 21 ng/mL, total FAEEs 344 ± 91 ng/mL. Detectability of FAEEs was found to be on the same time scale as BAC. In liquid blood samples containing ethanol, FAEE concentrations increase post-sampling. This study shows that the use of DBS fixation prevents additional FAEE formation in blood samples containing ethanol. Positive FAEE results obtained by DBS analysis can be used as evidence for the presence of ethanol in the original blood sample.

Interlaboratory comparison of phosphatidylethanol in dried blood spots using different sampling devices.

Phosphatidylethanol (PEth) has become an important marker to assess drinking behaviour and monitor abstinence. Despite its increasing use, knowledge on robustness and standardization and comparability of methods and results are still limited. In 2022, the first international consensus for the use of PEth and its interpretation was published. To establish an experience-based foundation for further harmonization, three rounds of interlaboratory comparison using microsamples were conducted. Participating laboratories sent their sampling devices to the laboratory of Forensic Toxicology at the University of Bern, where for each round, four different authentic blood samples were applied to the devices and sent back. The PEth 16:0/18:1 target concentrations covered a range between 16 and 474 ng/mL (0.023 and 0.676 µmol/L, respectively) and included sample concentrations close to the decision limits of 20 and 200 ng/mL (0.025 and 0.28 µmol/L, respectively). Evaluation of the results based on guidelines by Horwitz and the Society of Toxicological and Forensic Chemistry (GTFCh) showed that 73% of all participating laboratories quantified and reported all samples (N = 4 for each round) within the acceptable limits. More than 90% quantified and reported at least one sample within the acceptable limits.

Lyso-phosphatidylethanol detected by LC-MS/MS as a potential new marker for alcohol consumption.

Alcohol biomarkers are able to reflect the degree of recent or long-term alcohol consumption, covering different windows of detection. Phosphatidylethanols (PEths) are an emerging group of direct alcohol biomarkers that are widely applied in clinical and forensic applications. Their quantification can provide insight into an individual's drinking behaviour. Here, we present a new sub-class of yet unknown PEth species, LysoPEths, which are structurally related to PEth, but miss one fatty acyl chain. LysoPEths can be either a degradation product of PEth or a product of transesterification of lyso-phosphatidylcholine (LysoPC) with ethanol. To set up an analytical method, LysoPEth 16:0 was synthesised from PC 16:0/18:1 and characterised by LC-MS/MS, using an enzymatic method: phospholipase D (PLD), followed by phospholipase A2 (PLA2). Then, an LC-MS/MS method in MRM mode for LysoPEth 16:0 with additional LysoPEth species (LysoPEth 18:1, LysoPEth 18:2, and LysoPEth 20:4) and PEth 16:0/20:4 was developed. By incubation of freshly sampled venous blood of a teetotaller with ethanol at different concentrations, the formation of LysoPEth in parallel to PEth was investigated. With increasing ethanol concentrations, LysoPEth 16:0 was formed besides the known PEth species (PEth 16:0/18:1, PEth 16:0/18:2) for up to 72 h with LysoPEth concentrations being about three times lower than PEth concentrations. Storage of ethanol-free PEth-positive blood of an alcohol consumer at 37 °C showed that LysoPEth 16:0 concentrations increased, while PEth 16:0/18:1 concentrations decreased in the first 24 h for frozen/thawed blood, however not for freshly collected blood. Furthermore, LysoPEth 16:0 was detected in venous as well as lyophilised blood from clinical and forensic case work alongside with PEth 16:0/18:1, 16:0/18:2, and other PEth and LysoPEth species (PEth 16:0/20:4, LysoPEth 18:1, LysoPEth 18:2, and LysoPEth 20:4). LysoPEth 16:0 concentrations were found to be in linear correlation with PEth 16:0/18:1 (r2 = 0.75).

Application of Dried Urine Spots for Non-Targeted Quadrupole Time-of-Flight Drug Screening.

The use of dried urine spots (DUS) can simplify sample handling, shipment and storage when compared to liquid urine samples. To prepare DUS, a small amount of urine is pipetted on a filter paper card. The subsequent drying of the specimen can prevent the post-sampling formation or degradation of substances (e.g., caused by bacteria). To evaluate the potential of DUS screening, 17 authentic urine samples, containing a broad range of substances, were extracted and analyzed on a Sciex TripleTOF® 5600+ System using a non-targeted screening and library searching approach. The screening results were compared to the analysis of the same urine sample in liquid form, using the same high-resolution liquid chromatography--quadrupole time-of-flight mass spectrometry method. More than 65 different legal and illegal drugs were successfully identified within the investigated 17 urine samples using the DUS screening approach. When compared to the analysis of liquid urine, the following compounds could not be identified: 1x ecgonine methyl ester, 1x nicotine, 1x promazine and 1x 11-nor-9-carboxy-∆9-tetrahydrocannabinol. Overall, 95.2% of the target substances that have been detected in liquid urine were identified correctly using the DUS approach. In conclusion, DUS screening offers a simple, cost-effective and easier sample handling alternative to the traditional use of liquid urine and provides the detection of the most important substances for forensic requirements. Furthermore, the DUS sample preparation can be fully automated (sample documentation, internal standard application and extraction).

Fully automated dried blood spot sample handling and extraction for BoHV-1 antibody testing by ELISA.

This study is the first proof of concept of the DBS technology for Bovine alphaherpesvirus 1 (BoHV-1) antibody detection by ELISA after fully automated DBS extraction. DBS were prepared from nine BoHV-1 seropositive plasma samples spiked with erythrocytes. Spots were extracted automatically on a DBS-MS 500 HCT autosampler, as well as manually using a 3.2 mm puncher. DBS were equally prepared from 20 bovine seronegative EDTA-blood samples and extracted automatically. Extracts were tested in a commercial BoHV-1 antibody ELISA and results were compared with those from liquid plasma. Eight seropositive DBS samples were additionally tested in the ELISA after storage for four weeks at different conditions. After automated extraction all DBS samples yielded qualitatively correct results and were in full accordance with those obtained from liquid plasma. Automated extraction using a 6 mm extraction head was more sensitive than a 4 mm head. Stability of DBS was highest at - 20 °C and decreased with increasing temperature. Even after four weeks at 37 °C, most seropositive samples yielded a positive result in the ELISA. The minimal invasiveness, biosafety, and simplicity of DBS collection together with automated extraction represents an interesting, high-throughput compatible alternative to liquid blood samples for BoHV-1 monitoring or eradication programs.

Comparison of automated determination of phosphatidylethanol (PEth) in dried blood spots (DBS) with previous manual processing and testing.

Phosphatidylethanol (PEth) is a sensitive and specific biomarker of alcohol consumption in the prior 2-3 weeks. Standard, manual PEth testing using dried blood spots (DBS) is a multi-step time-consuming process. A novel, automated processing and testing method has been developed to decrease DBS processing and testing time. We conducted automated testing, using regioisomerically pure PEth reference material, on randomly selected DBS, which had previously been tested via manual methods and then stored for 3-6 years at -80 °C, to compare the results (PEth 16:0/18:1 homologue). We chose samples for re-testing using categories found in the literature as follows: 1) PEth <20 ng/mL; 2) PEth 20-200 ng/mL; 3) PEth >200-1000 ng/mL; 4) PEth >1000 ng/mL. We calculated agreement between the categories using the weighted kappa statistic (n = 49 DBS). We quantified agreement between continuous measures using the intraclass correlation coefficient (ICC), and further described the relationship between variables using Spearman correlation. The median PEth result was 155 ng/mL (interquartile range [IQR]: 1-1312 ng/mL) via automated methods and 98.8 ng/mL (IQR: 10.2-625.0 ng/mL) via manual methods. The weighted kappa comparing the automated to manual PEth results was 0.76 [95% Confidence Interval (CI): 0.66-0.86]. The ICC was 0.69 (95% CI: 0.54-0.79), and the Spearman correlation was 0.98 (95% CI: 0.95-0.99). While the new methods yielded somewhat higher PEth values, we found good to excellent agreement between clinically relevant PEth categories. Automated DBS processing and testing using new reference standards are promising methods for PEth testing.

Dried blood spots for anti-doping: Why just going volumetric may not be sufficient.

The perspective discusses quantitative DBS analysis for anti-doping testing in an athletic population and why only using volumetric sampling for this subgroup might not be enough. It presents examples to highlight where HCT variations occur, followed by a whole blood to plasma ratio and an HCT extraction bias discussion. Finally, options to correct for the HCT bias are presented.

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.

Fully automated dried blood spot sample handling and extraction for serological testing of SARS-CoV-2 antibodies.

At the beginning of 2020, an outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reached pandemic dimensions. Throughout the event, diagnostic tests function as an essential tool for understanding, mitigating, and implement strategies to curb and reduce infections. Here, we present a novel method for the fully automated dried blood spot (DBS) sample handling and extraction for serological testing of human IgG antibodies against SARS-CoV-2 using a commercial enzyme-linked immunosorbent assay (ELISA) testing kit. This proof-of-principle pilot study successfully demonstrates the recovery of antibodies in their intact form from DBS using automated, direct sample elution within 100 µl of extraction buffer. The use of minimally invasive DBS sampling provides an alternative to existing analytical procedures such as sampling by venipuncture or nasal swabs. Due to the ease of DBS collection, no third party need be involved, making at-home sampling possible (e.g., during quarantine).

Quantitative determination of phosphatidylethanol in dried blood spots for monitoring alcohol abstinence.

Phosphatidylethanol (PEth), which is formed by enzymatic reaction between ethanol and phosphatidylcholine, is a direct marker for alcohol usage. PEth has a long elimination half-life (~5-10 d) and specimens can be sampled using minimally invasive microsampling strategies. In combination with rapid analysis procedures PEth has proved to be advantageous for the detection of abstinence over other direct (e.g., ethyl glucuronide in blood, urine or hair) and indirect (e.g., carbohydrate-deficient transferrin in serum) alcohol markers. Although PEth determination is widely applied around the world, laboratory protocols are not standardized. Here we provide general guidelines for the analysis of PEth in dried blood spots (DBSs), including reference material evaluation, synthesis of a deuterated internal standard, preparation of calibration samples (reference material in teetotaller blood), and analyte separation and detection. The protocol contains information to extract the DBSs either manually or with a fully automated autosampler. Extraction of the analytes from DBS filter paper cards is performed using an organic extraction, followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). For accurate and reliable measurement of PEth, the two most abundant analogs, PEth 16:0/18:1 and PEth 16:0/18:2, are quantified. We show data that provide guidelines on how to interpret the results for both demographic studies and forensic applications. The described protocol can be applied by experienced laboratory staff with basic LC-MS/MS knowledge and takes 2 d to perform.

Variation in the Relative Isomer Abundance of Synthetic and Biologically Derived Phosphatidylethanols and Its Consequences for Reliable Quantification.

Phosphatidylethanol (PEth) in human blood samples is a marker for alcohol usage. Typically, PEth is detected by reversed-phase liquid chromatography coupled with negative ion tandem mass spectrometry, investigating the fatty acyl anions released from the precursor ion upon collision-induced dissociation (CID). It has been established that in other classes of asymmetric glycerophospholipids, the unimolecular fragmentation upon CID is biased depending on the relative position (known as sn-position) of each fatty acyl chain on the glycerol backbone. As such, the use of product ions in selected-reaction-monitoring (SRM) transitions could be prone to variability if more than one regioisomer is present in either the reference materials or the sample. Here, we have investigated the regioisomeric purity of three reference materials supplied by different vendors, labeled as PEth 16:0/18:1. Using CID coupled with ozone-induced dissociation, the regioisomeric purity (% 16:0 at sn-1) was determined to be 76, 80 and 99%. The parallel investigation of the negative ion CID mass spectra of standards revealed differences in product ion ratios for both fatty acyl chain product ions and ketene neutral loss product ions. Furthermore, investigation of the product ion abundances in CID spectra of PEth within authentic blood samples appears to indicate a limited natural variation in isomer populations between samples, with the cannonical, PEth 16:0/18:1 (16:0 at sn-1) predominant in all cases. Different reference material isomer distributions led to variation in fully automated quantification of PEth in 56 authentic dried blood spot (DBS) samples when a single quantifier ion was used. Our results suggest caution in ensuring that the regioisomeric compositions of reference materials are well-matched with those of the authentic blood samples.

The application of fully automated dried blood spot analysis for liquid chromatography-tandem mass spectrometry using the CAMAG DBS-MS 500 autosampler.

In the past decade, dried blood spot (DBS) sampling has been used increasingly for microsampling in various fields. This is predominantly driven by the significant advantages DBS offers regarding simple sample retrieval and shipment, combined with increased analyte stability. However, the manual handling of DBS samples is laborsome and prevents the use of a high-capacity bioanalytical workflow. The recent introduction of robotic DBS extraction systems in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has enabled the full automation of the analytical process. This results in overall higher sample throughput, minimal user interaction, and a significant reduction in consumables. Different instrumental setups are currently available which differ with respect to the extraction process, extract processing strategy, and internal standard application. This review article provides an overview of fully automated DBS analysis for one of these instruments, the DBS-MS 500 autosampler from CAMAG. The automated processes are described in detail and various applications are presented. Emphasis is placed on the advantages that the use of DBS, in combination with automation, brings - such as speed, reliability, and user-friendliness. Discussing DBS solutions for newborn screening, workplace drug testing, forensic screening, direct alcohol marker analysis, antiretroviral drugs, anti-epileptic drugs, and mass drug administration, the versatility and applicability of DBS are demonstrated in detail. In conclusion, this article shows how and why fully automated DBS analysis has penetrated the routine laboratory environment.

Fully Automated Optical Hematocrit Measurement From Dried Blood Spots.

The impact of the hematocrit (HCT) on the dried blood spot's (DBS) spreading area is one of the most important hurdles which prevents the full acceptance of quantitative microsampling strategies. Several destructive- and non-destructive strategies to assess the HCT from a DBS post-sampling have been presented. Unfortunately, the current methods are either labor-intensive, require a complicated algorithm, or are not automatable. Here, we present a novel setup that permits the fully automated reflectance analysis to measure the HCT from a DBS. The underlying principle is based on the concept presented by Capiau et al. for the non-destructive single-wavelength measurement of the HCT. The novel module was embedded within the DBS-MS 500 platform to enable high-throughput analysis of hematocrit values in combination with automated DBS extraction. The novel setup was assessed and optimized for the probe to card distance, stability, anti-coagulant, spotting volume, scan number, calibration variability, accuracy, and precision. It showed excellent inter-day (≤3.7%) and intra-day (≤1.16%) precision, as well as high accuracy when analyzing authentic samples 101%±7% (range:87%-127%). Besides, the simple and straightforward application of an HCT correction for DBS was demonstrated during a pharmacokinetic study with diclofenac involving three subjects. Thereby, the sample's HCT and the HCT impact on the analyte was assessed and compensated. In conclusion, the novel setup enables quantitative analysis of non-volumetric samples in an automated fashion without compromising the concept of cost-effective, minimally invasive sampling.

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 antidoping 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 antidoping 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.

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.