Automated Sample Preparation Method Research Articles

Quantitative Analysis of Lysergic Acid Diethylamide and Metabolite in Urine by Automated Extraction and Liquid Chromatography-Tandem Mass Spectrometry.

Recently, lysergic acid diethylamide (LSD) has become a resurgent drug of abuse. The detection of LSD is problematic because of the low dosage taken by users, light and heat sensitivity of the analyte and the lack of efficient analytical methods. Presented here is the validation of an automated sample preparation method for the analysis of LSD and its primary urinary metabolite, 2-oxo-3-hydroxy-LSD (OHLSD), in urine samples by liquid chromatography-tandem mass spectrometry. Analytes were extracted from urine using an automated Dispersive Pipette XTRaction method on Hamilton STAR and STARlet liquid handling systems. The limit of detection for both analytes was administratively defined at the lowest calibrator used in the experiments, and the limit of quantitation was 0.05 ng/mL for both analytes. All validation criteria were acceptable per Department of Defense Instruction 1010.16 requirements. This method offers an efficient, sensitive analytical solution to routinely evaluate large numbers of urine specimens for LSD in workplace drug deterrence programs.

High-Throughput Proteome Profiling of Plasma and Native Plasma Complexes Using Native Chromatography.

We describe a high-throughput method for co-fractionation mass spectrometry (CF-MS) profiling for native plasma protein profiling. CF-MS allows the profiling of endogenous protein complexes between samples. Proteins often interact with other proteins and form macromolecular complexes that are different in disease states as well as cell states and cell types. This protocol describes an example for the sample preparation of 954 individual size exclusion chromatography (SEC) fractions, derived from 18 plasma samples that were separated into 53 fractions. Eighteen plasma samples were chosen based on the TMTpro multiplexing, but this methodology can be adapted for fewer or larger numbers of samples as appropriate. Our automated sample preparation method allows for high-throughput native plasma profiling, and we provide detailed methods for both a label-free and an isobaric labeling approach, discuss the merits of each approach, and detail the advantages of combining these strategies for comprehensive native plasma proteome profiling.

SAMHSA drug panel screening in oral fluid: Development of screening method at 8 seconds per sample using LDTD-MS/MS

In 2019, the US Department of Health and Human Services (via the SAMHSA agency) has established scientific and technical guidelines for federal workplace drug testing programs in oral fluids (Federal Register/Vol. 84, No. 207, 2019). Screening various drug classes requires several different immunoassay reagents or an LC-MS/MS method with a longer analysis time per sample. LDTD-MS/MS technology combines the speed and the analysis of different drug classes with a single method. The goal of this presentation is to use an automated sample preparation method for LDTD-MS/MS screening of all compounds in a single operation. Quantisal collection devices were used in this method. Drug-free oral fluids of different volunteers were collected using the Quantisal device. After the collection of the oral fluid, the pad is transferred into a tube containing an extraction buffer. During this process, oral fluids are diluted by a factor of 3. The SAMHSA drug panel for an Oral fluid screen was spiked in extracts at a concentration around the decision point cut-off (Amphetamine: 50 ng/mL, Methamphetamine: 50 ng/mL, MDA: 50 ng/mL, MDMA: 50 ng/mL, PCP: 100 ng/mL, Morphine: 30 ng/mL, Hydromorphone: 30 ng/mL, Codeine: 30 ng/mL, Hydrocodone: 30 ng/mL, Cocaine: 30 ng/mL, Oxymorphone: 30 ng/mL, Oxycodone: 30 ng/mL, 6-AM: 4 ng/mL and THC: 4 ng/mL). Samples were extracted using the Azeo automated extraction system. The robot scans the barcode of the sample, generates a batch file for the LDTD-MS/MS system, extracts the sample and spots the LazWell plate. Salt assisted liquid-liquid extraction (SALLE) was used as the sample preparation method. LDTD (Phytronix Luxon) uses a carrier gas (air) flow rate of 6 L/min with water infusion at 20 uL/sec. Samples were desorbed from the surface of the plate using a laser ramp of 6 seconds to 65% power and 2 seconds hold at 65% power. Mass spectrometer (Shimadzu 8060) operates in positive MRM mode with a specific transition for the following drugs: Amphetamine, Methamphetamine, MDA, MDMA, PCP, Morphine, Hydromorphone, Codeine, Hydrocodone, Cocaine, Oxymorphone, Oxycodone, 6-AM and THC. Data acquisition systems of mass spectrometers were not designed to deal with signals of only a few seconds per sample. The synchronization sequence adds 6 to more than 15 seconds between each sample. To bypass this, all samples are acquired in a single file. To allow the analysis of such data, Cascade™ software is designed to detect, split, and integrate every sample peak acquired in a single file. Spiked samples around the decision point and blank solutions are used to validate the precision of the method. Each concentration must not exceed 20% CV and the mean concentration ± 2 times the standard deviation must not overlap with other concentrations at the decision point. The peak area against the IS ratio was used to normalize the signal. Replicate extractions are deposited on a LazWell plate and dried before analysis. No overlapping at the decision point is observed for all curves and the CV% was below 15% for within-run experiments. A dry and wet stability of sample extracts were performed. Precision criteria were applied. All results are within the acceptable criteria range. Oral fluids are collected from eight different volunteers. Samples are screened to verify the presence of each analyte (all samples were negative). Drugs are spiked at 50% cut-off (QC-L) and 200% cut-off (QC-H) and screened as unknown to verify the method sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy. A percentage of 100% was obtained for all validation parameters of all drugs. Luxon Ion Source® combined to a Shimadzu 8060 mass spectrometer system allows ultra-fast (8 seconds per sample) screening of a SAMHSA drug panel in oral fluid using a simple and automated sample preparation method.

Optimization of Automated Sample Preparation for Vitamin D Determination on a Biomek i7 Workstation

Vitamin D belongs to the fat-soluble vitamins and is an integral part of bone metabolism. In the human body, a decreased vitamin D level can be an additional risk factor for diseases like cancer, diabetes, and mental diseases. As a result, an enormous increase in the demand for vitamin D testing has been observed in recent years, increasing the demand for powerful methods for vitamin D determination at the same time.Automation is the key factor in increasing sample throughput. This study compares three fully automated sample preparation methods for the determination of 25(OH)D2 and 25(OH)D3 in plasma and serum samples. Starting from a semiautomated reference method, the method is tested manually and subsequently fully automated on the Biomek i7 Workstation by integrating a centrifuge and a positive pressure extractor into the workstation. Alternatively, the centrifugation for the separation of protein aggregates and supernatant is replaced by a filter plate. Finally, the sample throughput is further increased by using phospholipid removal cartridges. The results show that phospholipid removal significantly increases the recovery rates in liquid chromatography-mass spectrometry. With the phospholipid removal cartridges, recovery rates of 97.36% for 25(OH)D2 and 102.5% for 25(OH)D3 were achieved, whereas with the automated classic automated preparation method, the recovery rates were 83.31% for 25(OH)D2 and 86.54% for 25(OH)D3. In addition to the technical evaluation, the different methods were also examined with regard to their economic efficiency. Finally, the qualitative and quantitative performance of the developed methods is benchmarked with a selected semiautomatic reference method.

Validation of Solus One Salmonella from Select Food Matrixes and Stainless-Steel and Plastic Environmental Surfaces.

Background: Solus One Salmonella is designed to accurately detect Salmonella species (Salmonella enterica subspecies enterica, salamae, arizonae, diarizonae, houtenae, indica, and Salmonella bongori) from select food matrixes and stainless-steel and plastic environmental surfaces. Solus One Salmonella uses an antibody-based technology test system that is paired with media and our proprietary media supplement, the Solus One Salmonella supplement combined with a manual or automated sample preparation method. Objective: Solus One Salmonella was evaluated for inclusivity and exclusivity, and a matrix comparison study was done for six food matrixes (raw beef trim, pasteurized liquid egg, raw salmon, cheddar cheese, Romaine lettuce, nonfat dry milk) and two environmental surfaces (stainless steel and polystyrene). Methods: Solus One Salmonella was compared with the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 5: Salmonella (July 2018) and the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Manual, 4.09 (January 2017) in the matrix study. Both the manual and automated sample preparation methods were performed for cheddar cheese and stainless-steel environmental surfaces. Results: For the inclusivity and exclusivity evaluation, Solus One Salmonella correctly detected all 108 target organism isolates and correctly excluded all 35 nontarget strains that were analyzed. Conclusions: In the method comparison study, both Solus One Salmonella manual and automated sample preparation methods demonstrated no significant differences based on probability of detection (POD) statistical analysis between presumptive and confirmed results or between candidate and reference method results for the six food matrixes after 20-22 h and two environmental surfaces after 16-20 h of enrichment time. POD analysis of Solus One Salmonella method robustness, product consistency, and stability studies using the automated sample preparation method demonstrated no statistically significant differences.

An automated high-throughput sample preparation method using double-filtration for serum metabolite LC-MS analysis

A novel, automated sample preparation method for serum metabolite LC-MS analysis was developed using a double-filtration system.

Development of Universal, Automated Sample Acquisition, Preparation, and Delivery Devices and Methods for Pharmaceutical Applications

Continued emphasis in the pharmaceutical industry toward development of hybrid and continuous processes has led to a resurgence in process analytical technologies. The need to consistently and rapidly monitor the quality of material being made from these processes is a significant challenge that requires integrated, online analytics. Often the most challenging aspect of implementing real-time monitoring is ensuring that the analytical instrumentation is receiving a representative sample from the process. The Small Molecule Design and Development organization at Eli Lilly and Company has developed a highly adaptable process-to-analytics interface that has been broadly implemented across the development portfolio. The automated sample carts use only gravity and low-pressure nitrogen to obtain representative process samples, typically on the order of 0.3–6 mL. Subsequent sample preparation operations including quenching, derivatization, and most commonly dilution (10–250× is customary, but not restrictive) i...

A New Automated Method for the Analysis of Aromatic Amines in Human Urine by GC-MS/MS.

Cigarette smoking significantly increases the risk of cancer and cardiovascular diseases as well as premature death. Aromatic amines (AAs) such as o-toluidine, 2-aminonaphthalene and 4-aminobiphenyl are found in cigarette smoke and are well-established human bladder carcinogens presumably acting via the formation of DNA adducts. These amines may be metabolized in the liver to acetylated or glucuronidated forms or oxidized to a hydroxylamine which may react with protein and DNA to form adducts. Free, acetylated and glucuronidated AAs are excreted in urine and can be measured as exposure biomarkers. Using isotope dilution GC-MS/MS, our laboratory quantifies six urinary AAs that are known or suspected carcinogens-o-toluidine, 2,6-dimethylaniline, o-anisidine, 1-aminonaphthalene, 2-aminonaphthalene and 4-aminobiphenyl-for large population studies such as the National Health and Nutrition Examination Survey (NHANES). We also monitor two additional corresponding structural isomers-2-aminobiphenyl and 3-aminobiphenyl-to verify isomer separation. A new and improved automated sample preparation method was developed to quantify these AAs, in which, sample cleanup was done via Supported Liquid Extraction (SLE+ ISOLUTE®) on a Hamilton STAR™ workstation. This automated method increased sample throughput by reducing sample cleanup time from 8 to 4 h while maintaining precision (intra and inter-run coefficient of variation <7%) and accuracy (±17%). Recent improvements in our GC/MS method have enhanced our assay sensitivity and specificity, resulting in longer analytical column life and maintaining or reducing the limit of detection for all six analytes. Indigo ASCENTTM software (3.7.1, Indigo BioAutomation, Inc.) is used for peak integration, calibration and quantification. A streamlined sample data flow was created in parallel with the automated method, in which samples can be tracked from receiving to final laboratory information management system output with minimal human intervention, minimizing potential human error. This newly validated, automated method and sample data flow are currently applied in biomonitoring of AAs in the US noninstitutionalized population NHANES 2013-2014 cycle.

A New Automated Method and Sample Data Flow for Analysis of Volatile Nitrosamines in Human Urine.

Volatile nitrosamines (VNAs) are a group of compounds classified as probable (group 2A) and possible (group 2B) carcinogens in humans. Along with certain foods and contaminated drinking water, VNAs are detected at high levels in tobacco products and in both mainstream and sidestream smoke. Our laboratory monitors six urinary VNAs—N-nitrosodimethylamine (NDMA), N-nitrosomethylethylamine (NMEA), N-nitrosodiethylamine (NDEA), N-nitrosopiperidine (NPIP), N-nitrosopyrrolidine (NPYR), and N-nitrosomorpholine (NMOR)—using isotope dilution GC-MS/MS (QQQ) for large population studies such as the National Health and Nutrition Examination Survey (NHANES). In this paper, we report for the first time a new automated sample preparation method to more efficiently quantitate these VNAs. Automation is done using Hamilton STAR™ and Caliper Staccato™ workstations. This new automated method reduces sample preparation time from 4 hours to 2.5 hours while maintaining precision (inter-run CV < 10%) and accuracy (85% - 111%). More importantly this method increases sample throughput while maintaining a low limit of detection (<10 pg/mL) for all analytes. A streamlined sample data flow was created in parallel to the automated method, in which samples can be tracked from receiving to final LIMs output with minimal human intervention, further minimizing human error in the sample preparation process. This new automated method and the sample data flow are currently applied in bio-monitoring of VNAs in the US non-institutionalized population NHANES 2013-2014 cycle.

Bioanalytical laboratory automation development: why should we and how could we collaborate?

Bioanalytical laboratory automation development: why should we and how could we collaborate?

Acoustic trapping for bacteria identification in positive blood cultures with MALDI-TOF MS.

Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is currently changing the clinical routine for identification of microbial pathogens. One important application is the rapid identification of bacteria for the diagnosis of bloodstream infections (BSI). A novel approach based on acoustic trapping and an integrated selective enrichment target (ISET) microchip that improves the sample preparation step for this type of analysis is presented. The method is evaluated on clinically relevant samples in the form of Escherichia coli infected blood cultures. It is shown that noncontact acoustic trapping enables capture, enrichment, and washing of bacteria directly from the complex background of crude blood cultures. The technology replaces centrifugation-based separation with a faster and highly automated sample preparation method that minimizes manual handling of hazardous pathogens. The presented method includes a solid phase extraction step that was optimized for enrichment of the bacterial proteins and peptides that are used for bacterial identification. The acoustic trapping-based assay provided correct identification in 12 out 12 cases of E. coli positive blood cultures with an average score of 2.19 ± 0.09 compared to 1.98 ± 0.08 when using the standard assay. This new technology opens up the possibility to automate and speed up an important and widely used diagnostic assay for bloodstream infections.

Development of sample pretreatment approach and technology for peptidome

As a branch of proteomics, peptidome has been extensively applied in biomarker discovery, early diagnosis and pharmacy. In the research of peptidome, sample pretreatment plays a vital role. Here, we review the pretreatment approaches and techniques of peptidome, mainly including ultrafiltration, organic solvent precipitation, solid phase extraction and so on. The biggest challenge of the development of peptidome is the large number of high-abundance proteins in the sample. Therefore, there is an urgent need to develop the fast, efficient, high-throughput and automated sample preparation methods and techniques.

Abstract LB-230: Automating sample preparation for single-cell protein phosphorylation analysis by flow cytometry

Abstract Protein phosphorylation plays essential roles in regulating cellproliferation, differentiation and other cellular processes. Aberrant regulation of protein kinases, can drive malignancy through inappropriate cell growth and survival signals. Understanding the phosphorylation status of specific cellular proteins in cancer has proven valuable in developing targeted therapies and assays to identify specific drug responses in individual cancer patients. The ability to analyze cells at the single cell level allows flow cytometry to measure protein phosphorylation in rare subsets of cells in complex populations such as blood or bone marrow specimens. However, sample preparation for protein phosphorylation analysis by flow cytometry can be technically challenging, time-consuming, and subject to error when performed manually. Furthermore, the procedure demands strict control of experimental conditions such as time and temperature due to the dynamic nature of protein phosphorylation. In this report, we describe an automated sample preparation method using a @Biomek® NXP Span-8 work-station to monitor the phosphorylation status of ERK and p38 MAPK in human peripheral blood monocytes. The method allows automation of sample preparation steps, including 1) ERK/p38 MAPK stimulation, 2) fixation, 3) erythrocyte lysis, 4) cell permeabilization, and, 5) staining with phospho-specific and cell surface marker antibodies. Using this approach, we were able to observe p38 MAPK and ERK activation in CD14+ monocytes following lipopolysaccharide (LPS) or phorbol-12-myristate-13 acetate (PMA) treatment. The automated sample preparation workstation is capable of processing 48 samples in approximately 2.5 hours. Note: *Method cited Not for Clinical purposes. @For Laboratory Use Only; not for use in diagnostic procedures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-230.

Abstract 2220: Analyzing cancer with single cell resolution using droplet technology

Abstract Cancers develop and grow in the complex environments present in normal tissue, and tumor cells are composed of heterogeneous mixtures of both clonal transformed cells and other cell types. We demonstrate the use of microfluidic-generated single cell microdroplets in workflows that capture both phenotypic and genomic information from individual cells, enabling analysis of tumors with single cell resolution. This transformative new tool will allow for more thorough examinations of the variations that influence the predisposition and composition of cancers, and the cellular responses to therapeutic and prevention agents. Uniform picoliter-nanoliter volume aqueous droplets suspended in an immiscible oil provide unique opportunities for fluorescence-based analytical techniques, including sorting relevant sub-populations at rates of several hundred cells per second, coupled directly to single cell genomic characterization. We use a defined set of control samples to perform low-bias single cell whole genome amplification (WGA) within the sorted microdroplets, and show how following this with droplet PCR-based targeted amplification (of many thousands of selected genomic loci using a cancer-focused droplet primer library) provides an automated sample preparation method for any Next Gen sequencing platform. Single cell microdroplet technology results in a cost-effective method to gain sequence information from phenotypically selected individual cells, addressing cancer gene discovery and validation by powering the larger studies required to classify driver or passenger mutations and analyzing how epigenomic features (such as cytosine methylation) regulate transcriptional changes that can underlie cancer development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2220.

Automated sample preparation method for mass spectrometry analysis on recombinant proteins

A completely automated procedure for the purification and desalting of proteins with a polyhistidine purification tag prior to mass spectrometry analysis is presented. The system is ideal for rapid quality control and optimization studies and it provides researchers with a straightforward, reliable tool for studies of recombinant proteins. Forty-eight samples can be prepared within 4.5 h and only small cultivation and buffer volumes are needed. In this proof of concept, 19,000–35,000 Da recombinant proteins from both crude and clarified cell lysates were successfully prepared for subsequent analysis by electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry as well as by gel electrophoresis.

A new automated method for isolation of Chlamydia trachomatis from urine eliminates inhibition and increases robustness for NAAT systems

Chlamydia trachomatis is a leading cause of sexually transmitted infection. Diagnostic methods with easy non-invasive sample collection are important to increase testing and hence to reduce the spread of this infection. To enable more use of urine samples in C. trachomatis diagnostics, automation is an absolute requirement since obtaining high-quality DNA from urine specimens involves extensive processing. Here, we present a study in which a new automated sample preparation method, BUGS'n BEADS™ STI (BnB STI), was used up-front of the BDProbeTec™ ET end point analysis and compared with the full BDProbeTec™ ET method to analyze C. trachomatis in 1002 urine samples. The BnB STI system represents a new concept within magnetic sample preparation in which bacteria are first isolated from the sample material followed by purification of bacterial nucleic acid using the same magnetic particles. Similar sensitivity and specificity were obtained with both methods. None of the samples processed with BnB STI inhibited the BDProbeTec™ ET test whereas 1.8% showed inhibition when processed according to the manual BDProbeTec™ ET DNA preparation method. Moreover, the average MOTA scores obtained with the BnB STI system were 48% higher for all amplification controls and 57% higher for positive samples, compared to the manual sample preparation. Based on these results and the significant reduction in hands-on-time for urine sample processing, the automated BnB STI sample preparation method was implemented for routine analysis of C. trachomatis from urine samples.

Automated Target Preparation for Microarray-Based Gene Expression Analysis

DNA microarrays have rapidly evolved toward a platform for massively paralleled gene expression analysis. Despite its widespread use, the technology has been criticized to be vulnerable to technical variability. Addressing this issue, recent comparative, interplatform, and interlaboratory studies have revealed that, given defined procedures for "wet lab" experiments and data processing, a satisfactory reproducibility and little experimental variability can be achieved. In view of these advances in standardization, the requirement for uniform sample preparation becomes evident, especially if a microarray platform is used as a facility, i.e., by different users working in the laboratory. While one option to reduce technical variability is to dedicate one laboratory technician to all microarray studies, we have decided to automate the entire RNA sample preparation implementing a liquid handling system coupled to a thermocycler and a microtiter plate reader. Indeed, automated RNA sample preparation prior to chip analysis enables (1) the reduction of experimentally caused result variability, (2) the separation of (important) biological variability from (undesired) experimental variation, and (3) interstudy comparison of gene expression results. Our robotic platform can process up to 24 samples in parallel, using an automated sample preparation method that produces high-quality biotin-labeled cRNA ready to be hybridized on Affymetrix GeneChips. The results show that the technical interexperiment variation is less pronounced than with manually prepared samples. Moreover, experiments using the same starting material showed that the automated process yields a good reproducibility between samples.

Suitability of tandem-in-time mass spectrometry for polybrominated diphenylether measurement in fish and shellfish samples: Comparison with high resolution mass spectrometry

The first part of the present study focused on the development of an alternative automated sample preparation method for the measurement of selected polybrominated diphenyl ethers (PBDEs) in fish and shellfish. A previously developed automated method has been further optimized and simplified to decrease blank levels and cost. Sorbent and solvent quantities have been significantly reduced without altering extract quality. The second part of the study consisted of comparing tandem-in-time quadrupole ion storage mass spectrometry (QISTMS/MS) to high resolution mass spectrometry (HRMS) for the isotope dilution (ID) measurement step after gas chromatography (GC) separation. Both mass spectrometric methods performed similarly in terms of accuracy but better precision was observed for HRMS. Although better sensitivity can be attained with the high resolution sector instrument, method limits of quantification (mLOQs) were very similar for both approaches as they were dependent on the procedural blanks levels. The mLOQ values ranged between 0.04 and 3.56 ng/g fat, depending on the congener. They allowed the unambiguous identification and quantification of all target analytes, except for BDE-183, in most considered fish extracts. An analytical procedure based on rapid automated sample preparation and QISTMS/MS appeared to be suitable for the measurement of PBDEs in fish and shellfish speciment under quality assurance/quality control (QA/QC) criteria.

Incorporating Automation in Undergraduate Laboratories

Automated techniques and philosophies, which have become increasingly important in modern laboratories, are not traditionally covered in undergraduate chemistry curricula. To this end, we have been incorporating automated sample preparation methods using standard robotic workstations in our undergraduate analytical chemistry laboratory. Using a Benchmate™ II Workstation, an automated method has been developed and implemented for the solid phase extraction of capsaicins from commercial hot pepper sauces prior to liquid chromatographic analysis. This paper reports on pedagogical aspects of incorporating automation into the undergraduate curriculum as well as results obtained for manual and automated extractions conducted by students.

Incorporating Automation in Undergraduate Laboratories

Automated techniques and philosophies, which have become increasingly important in modern laboratories, are not traditionally covered in undergraduate chemistry curricula. To this end, we have been incorporating automated sample preparation methods using standard robotic workstations in our undergraduate analytical chemistry laboratory. Using a Benchmate™ II Workstation, an automated method has been developed and implemented for the solid phase extraction of capsaicins from commercial hot pepper sauces prior to liquid chromatographic analysis. This paper reports on pedagogical aspects of incorporating automation into the undergraduate curriculum as well as results obtained for manual and automated extractions conducted by students.