On the complex relationship between resilience and hair cortisol levels in adolescence despite parental physical abuse: a fourth wave of resilience research.

Introduction: To understand the family's role in adolescents' mental health development and the connection to neurodevelopmental disorders related to experienced parental physical abuse, we first explored resilience pathways longitudinally and secondly, connected the identified patterns to adolescents' hair cortisol levels that are rooted in the hypothalamic-pituitary-adrenal axis as the main stress response system and connected brain structure alterations. Methods: We analyzed longitudinal online questionnaire data for three consecutive high school years (from seventh to ninth grade) and four survey waves from a representative sample of n = 1609 high school students in Switzerland on violence-resilience pathways. Furthermore, we collected students' hair samples from a subsample of n = 229 at survey wave 4. About 30% of the participating adolescents had been physically abused by their parents. Out of the overall sample, we drew a subsample of adolescents with parental abuse experiences (survey wave 1 n = 509; survey wave 2 n = 506; survey wave 3 n = 561; survey wave 4 n = 560). Results: Despite the odds, about 20-30% of adolescents who have experienced parental physical abuse escaped the family violence cycle and can be called resilient. By applying a person-oriented analytical approach via latent class and transition analysis, we longitudinally identified and compared four distinct violence-resilience patterns. We identified violence resilience as a multidimensional latent construct, which includes hedonic and eudaimonic protective and risk indicators. Because resilience should not solely be operationalized based on the lack of psychopathology, our latent construct included both feeling good (hedonic indicators such as high levels of self-esteem and low levels of depression/anxiety and dissociation) and doing well (eudaimonic indicators such as high levels of self-determination and self-efficacy as well as low levels of aggression toward peers). Discussion: The present study confirmed that higher cortisol levels significantly relate to the comorbid pattern (internalizing and externalizing symptoms), and further confirmed the presence of lasting alterations in brain structures. In this way, we corroborated the insight that when studying the resilience pathways and trajectories of abused adolescents, biological markers such as hair cortisol significantly enhance and deepen the understanding of the longitudinal mechanisms of psychological markers (e.g., self-determination, self-esteem, self-efficacy) that are commonly applied in questionnaires.

Analysing and quantifying chronic stress-associated endogenous steroids in hair samples.

In previous studies, various steroids have been associated with stress and have therefore been quantified to investigate stress-related questions. Since the main stress-related steroid cortisol follows a circadian rhythm, often hair is analysed to quantify this steroid. Further, hair analysis gives the unique possibility of long-time monitoring by analysing a certain segment of hair, since hair grows on average 1 cm per month. Hair is a difficult matrix due to the complex sample preparation with many steps including washing and grinding, followed by various extraction steps. Additionally, steroids are endogenous and are therefore present in the hair matrix. Hence, no analyte free matrix is available, which is needed for the quantification via external calibrators. To overcome this problem, the so-called surrogate methods can be used, for which a 13 C3 labelled or deuterated reference compound of the steroid of interest is used for quantification. In the present study, a surrogate method was developed and fully validated for the quantitative analysis of seven steroids in human hair. Validation experiments showed that the method is further suitable for semi-quantitative analysis of estradiol. However, it is not suitable for the analysis of androsterone and DHEAS. The method was successfully used to analyse steroids in a comprehensive study of 360 adolescent hair samples, enabling research into stress markers.

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.

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.

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.

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.

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.

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 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).

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.

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 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.

Development and validation of an LC-MS/MS method for the analysis of ivermectin in plasma, whole blood, and dried blood spots using a fully automatic extraction system.

Ivermectin is deployed in mass drug administration (MDA) campaigns to control parasitic diseases in the tropics, with billions of treatments having been administered in the last three decades. Simple blood sampling tools, like the dried blood spots (DBS) technique, are needed to monitor treatments in such challenging settings. Thus, we developed a fully automated method for the analysis of ivermectin in DBS microsamples, including a bioanalytical and clinical validation. Automated extraction was carried out using a DBS-MS 500 autosampler which was coupled to a LC-MS/MS system. DBS were extracted with 20 µL solvent and eluted on a C8 analytical column. Analysis was performed by multiple reaction monitoring in the positive mode. Automated DBS extraction resulted in consistent recoveries (62.8 ± 4.3%) and matrix effects (68.0 ± 8.1%) between different donors and concentration levels. Intra- and inter-day accuracy and precision deviations were ≤15%, while samples with hematocrits from 20 to 60% could be quantified reliably. The achieved sensitivity of 1 ng/mL in DBS samples is sufficient to analyze ivermectin at the dose given (single oral administration of 12 mg) over a period of at least 72 h post treatment. Importantly, DBS samples are stable after one-month storage at room temperature (accuracy: 88.8-96.2%), thus samples collected in the field must not be shipped on dry ice. Ivermectin concentrations in venous and capillary blood agreed strongly, with a mean difference of -4.8%. Moreover, the drying process of DBS did not alter the analysis and importantly plasma concentrations can be estimated from DBS data using the hematocrit and red blood cell partitioning as correction factor. Our method enables uncomplicated sample collection and shipment as well as automated analysis of large amounts of samples, which is key to surveying MDA campaigns in remote settings.

Fully Automated Forensic Routine Dried Blood Spot Screening for Workplace Testing.

In this study, we describe the transfer of a new and fully automated workflow for the cost-effective drug screening of large populations based on the dried blood spot (DBS) technology. The method was installed at a routine poison control center and applied for DBS and dried urine spot (DUS) samples. A fast method focusing on the high-interest drugs and an extended screening method were developed on the automated platform. The dried cards were integrated into the automated workflow, in which the cards were checked in a camera recognition system, spiked with deuterated standards via an in-built spraying module and directly extracted. The extract was transferred online to an analytical LC column and then to the electrospray ionization tandem mass spectrometry system. The target compounds were analyzed in positive multiple-reaction monitoring mode. Before each sample batch or analysis day, calibration samples were measured to balance inter-day variations and to avoid false negative samples. An internal standard was integrated prior the sample extraction to allow in process control. A total of 28 target compounds were analyzed and directly extracted within 5 min per sample. This fast screening method was then extended to 20 min, enabling the usage of a Forensic Toxicology Database to screen over 1,200 drugs. The method gives confident positive/negative results for all tested drugs at their individual cut-off concentration. Good precision (±15%, respectively ±20% at limit of quantification) and correlation within the calibration range from 5 to 1,000 ng/mL was obtained. The method was finally applied to real cases from the lab and cross-checked with the existing methodologies.

Using dried blood spots to facilitate therapeutic drug monitoring of antiretroviral drugs in resource-poor regions.

Objectives: We evaluated whether dried blood spots (DBS) are suitable to monitor combined ART when samples are collected in rural Tanzania and transported over a long distance to a specialized bioanalytical laboratory. Methods: Plasma and DBS samples were collected in Tanzania from study patients treated with nevirapine, efavirenz or lopinavir. In addition, plasma, whole blood and DBS samples were obtained from a cohort of HIV patients at the site of the bioanalytical laboratory in Switzerland. DBS samples were analysed using a fully automated LC-MS/MS method. Results: Comparison of DBS versus plasma concentrations of samples obtained from the bridging study in Switzerland indicated an acceptable bias only for nevirapine (18.4%), whereas for efavirenz and lopinavir a pronounced difference of -47.4% and -48.1% was found, respectively. Adjusting the DBS concentrations by the haematocrit and the fraction of drug bound to plasma proteins removed this bias [efavirenz +9.4% (-6.9% to +25.7%), lopinavir +2.2% (-20.0% to +24.2%)]. Storage and transportation of samples from Tanzania to Switzerland did not affect the good agreement between plasma and DBS for nevirapine [-2.9% (-34.7% to +29.0%)] and efavirenz [-9.6% (-42.9% to +23.8%)]. For lopinavir, however, adjusted DBS concentrations remained considerably below [-32.8% (-70.4% to +4.8%)] corresponding plasma concentrations due to decay of lopinavir in DBS obtained under field conditions. Conclusions: Our field study shows that the DBS technique is a suitable tool for therapeutic drug monitoring in resource-poor regions; however, sample stability remains an issue for certain analytes and therefore needs special consideration.

Extended and Fully Automated Newborn Screening Method for Mass Spectrometry Detection.

A new and fully automated newborn screening method for mass spectrometry was introduced in this paper. Pathological relevant amino acids, acylcarnitines, and certain steroids are detected within 4 min per sample. Each sample is treated in an automated and standardized workflow, where a mixture of deuterated internal standards is sprayed onto the sample before extraction. All compounds showed good linearity, and intra- and inter-day variation lies within the acceptance criteria (except for aspartic acid). The described workflow decreases analysis cost and labor while improving the sample traceability towards good laboratory practice.