Analytical Method For Separation Research Articles

Robust analytical method for the enantiomeric separation of lurasidone hydrochloride: Integrating analytical quality by design and green chemistry principles

Schizophrenia, affecting approximately 1% of the global population, necessitates effective treatments like Lurasidone (LUR). However, the accurate separation of LUR from its enantiomeric impurities remains a significant challenge in pharmaceutical analysis. This study addresses this issue by developing and validating an optimized HPLC method for the simultaneous determination of LUR and its enantiomeric impurity using Analytical Quality by Design (AQbD) principles. The separation was achieved on a Chiralcel OD-H column with isocratic elution employing a hexane-ethanol mixture (92:8, v/v) at a flow rate of 0.9 mL/min, UV detection at 215 nm, and a column temperature of 32 °C. The method was validated using accuracy profiles. Environmental sustainability was assessed using various Green Analytical Chemistry (GAC) metrics, demonstrating favorable greenness attributes. This methodological framework not only enhances pharmaceutical quality control but also promotes sustainability in analytical practices, supporting its application in drug manufacturing and regulatory compliance.

Technical Note: A technique to convert NO2 to NO2− with S(IV) and its application to measuring nitrate photolysis

Abstract. Nitrate photolysis is a potentially significant mechanism for “renoxifying” the atmosphere, i.e., converting nitrate into nitrogen oxides – nitrogen dioxide (NO2) and nitric oxide (NO) – and nitrous acid (HONO). Nitrate photolysis in the environment occurs through two channels which produce (1) NO2 and hydroxyl radical (⚫OH) and (2) nitrite (NO2-) and an oxygen atom (O(3P)). Although the aqueous quantum yields and photolysis rate constants of both channels have been established, field observations suggest that nitrate photolysis is enhanced in the environment. Laboratory studies investigating these enhancements typically only measure one of the two photo-channels, since measuring both channels generally requires separate analytical methods and instrumentation. However, measuring only one channel makes it difficult to assess whether secondary chemistry is enhancing one channel at the expense of the other or if there is an overall enhancement of nitrate photochemistry. Here, we show that the addition of S(IV), i.e., bisulfite and sulfite, can convert NO2 to NO2-, allowing for measurement of both nitrate photolysis channels with the same equipment. By varying the concentration of S(IV) and exploring method parameters, we determine the experimental conditions that quantitatively convert NO2 and accurately quantify the resulting NO2-. We then apply the method to a test case, showing how an ⚫OH scavenger in solution prevents the oxidation of NO2- to NO2 but does not enhance the overall photolysis efficiency of nitrate.

Study on the combined analytical method for separation of uranium and plutonium by TEVA+DGA and TEVA+UTEVA resin ICP-MS

Study on the combined analytical method for separation of uranium and plutonium by TEVA+DGA and TEVA+UTEVA resin ICP-MS

Separation of full and empty adeno-associated virus capsids by anion-exchange chromatography using choline-type salts

Development of clinically desirable adeno-associated virus (AAV) vectors with optimal genome design requires rapid and accurate analytical methods to assess AAV quality. Anion-exchange (AEX) chromatography provides a powerful analytical method for full/empty AAV capsid ratio determination. However, the current AEX methodology for separation of empty and full AAV capsids largely relies on the use of the highly toxic tetramethylammonium chloride (TMAC). Here, we describe a novel analytical AEX method for separation of empty and full AAV capsids that uses only non-toxic, choline-type compounds that contain structural similarity to the quaternary ammonium ligand present on the surface of AEX resin. Choline-Cl gradient, combined with sensitive fluorescence detection, allowed a safe and effective separation of empty and full AAV capsids with reproducible empty/full ratio determination. The choline-based assay was suitable for commonly used serotypes, AAV2, AAV5, AAV6, and AAV8. The limit of detection was ∼3.9 × 108 virus particles in the assay. A gradient-hold step-gradient elution with choline-Cl resulted in enhanced baseline separation of empty and full AAV8 capsids. In summary, the use of choline-Cl in the AEX assay is recommended for empty/full capsid ratio determination and other applications in AAV production, and it eliminates the necessity of using toxic TMAC.

Medicinal Importance, Pharmacological Activity and Analytical Aspects of Flavonoid 'Irisflorentin' from Belamcanda chinensis (L.) DC.

Flavonoids are an important class of phytochemicals found to be present in plants and their derived products. Belamcanda chinensis (L.) DC has been used in Traditional Chinese medicine for multiple therapeutic purposes for a very long time. Belamcanda chinensis have been reported to have antipyretic, antidote, expectorant, antiphlogistic, and analgesic activity in the scientific fields. Scientific information on irisflorentin for its medicinal importance and pharmacological activity has been collected through electronic search engines, including Science Direct, Google, Google Scholar, PubMed, and Scopus, and analyzed in the present investigation in order to know the biological potential of irisflorentin. However, analytical aspects of irisflorentin are also discussed in the present work. Further detailed pharmacological activity data of irisflorentin have been collected and analyzed in the present work in order to know the therapeutic potential of irisflorentin in medicine. Scientific data analysis of different research work signified the biological importance and therapeutic benefit of Belamcanda chinensis and irisflorentin in medicine. Scientific data analysis revealed the biological effectiveness of irisflorentin against Parkinson's disease, inflammation, cancer, and brain disorders. Further scientific data analysis also signified the biological potential of irisflorentin for its α-glucosidase inhibitory potential and immunotherapeutic adjuvant in medicine. Metabolism and pharmacokinetic parameters have also been discussed in the present work. Analytical data showed the importance of various analytical methods for separation, identification and estimation of irisflorentin in different biological samples. Scientific data analysis of various research works signified the biological potential and therapeutic effectiveness of irisflorentin in medicine. However, clinical data of irisflorentin should be also investigated in order to know their pharmacokinetic parameters and safety issue in medicine.

Quantification of hetero-cyclic amines from different categories of braised beef by optimized UPLC-TQ-XS/ESI method.

This research work has developed and optimized a sensitive analytical method for separation and quantification of heterocyclic amines (HCAs) mainly including PhIP, Harman, Norharman, IQ, MeIQ, AαC, MeAαC and Trp-P-2 by optimizing UPLC-TQ-XS using electrospray ionization source (ESI+) on ACQUITY UPLC® BEH C18 column in <7min, from braised beef sample matrix. Meanwhile, modified HCAs extraction by modifying QuEChERS (quick, easy, cheap, efficient, rugged and safe) technique and revisited with solid phase extraction (SPE) for HCAs purification, instead using traditional QuEChERS salts. Moreover, optimized pH conditions of HCA extracts before purification, for better extraction recoveries. Furthermore, this method was validated in terms of method validation parameters. Lastly, simulation of real braised beef model provided the minimum formation of HCAs by optimizing cooking parameters and precursors in a cooking system. Therefore, this method could be applied simultaneously on braised beef matrix either marketed or home cooked for HCAs analysis.

A LC-MS/MS method for determination of clenbuterol enantiomers in animal tissues and its application to the enantioselective distribution in Bama mini-pigs

ObjectivesTo establish and validate a simple and reliable analytical method for separation and determination of clenbuterol enantiomers (R-(-)-clenbuterol &S-(+)-clenbuterol) in animal tissues, and apply it to the enantioselective distribution of clenbuterol in Bama mini-pigs. MethodsA LC-MS/MS analytical method was developed and validated in positive multiple reaction monitoring mode with electrospray ionization. After perchloric acid deproteinization, samples were pretreated only by one step liquid–liquid extraction using tert-butyl methyl ether under strong alkaline condition. Teicoplanin was used as chiral selector and 10 mM ammonium formate methanol solution was used as mobile phase. The optimized chromatographic separation conditions were completed in 8 min. Two chiral isomers in 11 edible tissues from Bama mini-pigs were investigated. ResultsR-(-)-clenbuterol and S-(+)-clenbuterol can be baseline separated and accurately analyzed with a linear range of 5–500 ng/g. Accuracies ranged from −11.9–13.0% for R-(-)-clenbuterol and −10.2–13.2% for S-(+)-clenbuterol, intra-day and inter-day precisions were between 0.7 and 6.1% for R-(-)-clenbuterol and 1.6–5.9% for S-(+)-clenbuterol. R/S ratios in edible tissues of pigs were all significantly lower than 1. ConclusionsThe analytical method has good specificity and robustness in determination of R-(-)-clenbuterol and S-(+)-clenbuterol in animal tissues, and can be used as a routine analysis method for food safety and doping control. There is a significant difference in R/S ratio between pig feeding tissues and pharmaceutical preparations (racemate with R/S ratio of 1), which makes it possible to identify the source of clenbuterol in doping control and investigation.

Analytical separation of plasmid DNA isoforms using anion exchanging chromatographic monoliths with 6µm channels.

High-performance liquid chromatography (HPLC)-based analytical assays are used to effectively monitor purity and quantity of plasmid DNA (pDNA) throughout the purification process. However, the phenomenon of physical entrapment of open circular (OC) isoforms pDNA inside narrow channels of chromatographic support decreases its accuracy and precision and the effect increases with pDNA size. The purpose of the study was to develop a chromatographic method for accurate analytical separation between isoforms of <16kbp pDNA using weak anion exchanging monolithic column with large (6µm) convective channels. Purified samples of 4.7 and 15.4kbp large pDNA with known isoform composition were prepared and their isoforms separated in ascending salt gradient. Both OC and supercoiled (SC) isoforms were baseline separated at a flow rate below 0.5mLmin-1 in a guanidinium chloride (GdnCl) gradient with a ≥95% OC pDNA elution recovery. However, these chromatographic conditions increased 2 times the peak width for linear (LIN) pDNA isoform compared to the results using monoliths with 1.4µm channel size. If other chaotropic agents, such as urea or thiocyanate (SCN), were added to Gdn ions, the elution volume for LIN isoform decreased. Optimization of combined GdnCl/GdnSCN gradient for pDNA elution resulted in a simple and robust chromatographic method, where OC-LIN and LIN-SC pDNA (up to 15kbp size) were separated with resolution above 1.0 and above 2.0, respectively. The accessibility and general acceptance of anion exchange chromatography for pDNA analytics give the newly developed method a great potential for in-process control monitoring of pDNA production processes.

Integrated Analysis of Polycyclic Aromatic Hydrocarbons and Polychlorinated Biphenyls: A Comparison of the Effectiveness of Selected Methods on Dried Fruit Matrices

Polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) are groups of chemical substances commonly found in the environment. Because of large differences in the concentrations of PAHs and PCBs in the materials tested, separate analytical methods specific to each group of compounds are usually used. The aim of this study was to compare methods for the determination of PAHs and PCBs that permit the simultaneous determination of these compounds from one solvent extract. The analysis of the content of 15 PCB congeners and 16 PAHs was conducted using dried fruits. The analyses were performed with gas chromatography coupled with mass spectrometry. PAHs and PCBs were determined separately in each fruit sample using specific extraction and cleanup procedures for the respective groups of compounds. Analyses were also performed with two methods that permitted the simultaneous analysis of PAHs and PCBs in one solvent extract. The integrated methods did not provide adequate extract cleanup of interfering substances; consequently, the results of determinations of PAHs and PCBs using these methods were significantly different from the values obtained with proven determination methods for PAHs and PCBs.

Green Analytical Method for Simultaneous Determination of Glucosamine and Calcium in Dietary Supplements by Capillary Electrophoresis with Capacitively Coupled Contactless Conductivity Detection.

The need for analytical methods that are fast, affordable, and ecologically friendly is expanding. Because of its low solvent consumption, minimal waste production, and speedy analysis, capillary electrophoresis is considered a "green" choice among analytical separation methods. With these "green" features, we have utilized the capillary electrophoresis method with capacitively coupled contactless conductivity detection (CE-C4D) to simultaneously determine glucosamine and Ca2+ in dietary supplements. The CE analysis was performed in fused silica capillaries (50 μm inner diameter, 40 cm total length, 30 cm effective length), and the analytical time was around 5 min. After optimization, the CE conditions for selective determination of glucosamine and Ca2+ were obtained, including a 10 mM tris (hydroxymethyl) aminomethane/acetic acid (Tris/Ace) buffer of pH 5.0 as the background electrolyte; separation voltage of 20 kV; and hydrodynamic injection (siphoning) at 25 cm height for 30 s. The method illustrated good linearity over the concentration range of 5.00 to 200 mg/L of for glucosamine (R 2 = 0.9994) and 1.00 to 100 mg/L for Ca2+ (R 2 = 0.9994). Under the optimum conditions, the detection limit of glucosamine was 1.00 mg/L, while that of Ca2+ was 0.05 mg/L. The validated method successfully analyzed glucosamine and Ca2+ in seven dietary supplement samples. The measured concentrations were generally in line with the values of label claims and with cross-checking data from reference methods (HPLC and ICP-OES).

Benchmarking polymeric cryogels for immobilized metal affinity chromatography

Cryogels are polymers prepared in frozen environment and seen as new separation matrices with their applications in many bioseparation methods. They have significant benefits including supermacroporosity, short diffusion path, low pressure, and low resistance to both adsorption and elution. Macro and connected pores give cryogels a unique spongy structure. Immobilized metal affinity chromatography (IMAC) is a generally employed analytical separation method for the purification of biomolecules. Several transition ions create stable complexes with electron-rich compounds. IMAC sorbent is obtained by complexing first-order transition metal ions over chelating agents. Lysozyme is an enzyme found in various vertebrate cells and secretions. Common applications include its use as a cell disrupting agent, as an anti-bacterial agent, as a food additive, and as a medicine for the treatment of infections and ulcers. In this study, cryogel-based polymeric material prepared by free-radical polymerization method with hydroxyethyl methacrylate/glycidyl methacrylate monomer pair were covalently interacted with iminodiacetic acid metal chelating agent, and regions showing affinity for lysozyme enzyme were formed by binding with Ni(II) ions. The cryogel was first characterized by Fourier transform infrared spectrophotometer, scanning electron microscopy, thermal gravimetric analysis, X-ray photoelectron spectroscopy and swelling test. Then, the effects of pH, lysozyme concentration, temperature, ionic strength and flow rate on lysozyme adsorption capacity were explored and optimum conditions were found.

Rapid Analysis of Plasmalogen Individual Species by High-Resolution Mass Spectrometry.

Analysis and quantification of ether-lipid phospholipid species-also known as plasmalogens-is a crucial step in the study of the biological functions played by these lipids. Application of analytical separation methods and high-resolution mass spectrometry has gained much attention in this regard, while resolution issues and time-consuming sequences interfered with these advances. Herein, we describe a simple and rapid method for the analysis of plasmalogen (Pl) species by HILIC-HRMS. This method is able to identify and quantify relative levels of ethanolamine-plasmalogens (PlsEtn) and choline-plasmalogens (PlsCho) in biological matrices such as whole blood, plasma, erythrocytes, and also retina. Moreover, we provide a detailed and modified lipid extraction method that is applicable to almost all biological matrices.

Shape-Enhanced Open-Channel Hydrodynamic Chromatography.

Hydrodynamic chromatography (HDC) is a well-established analytical separation method for the size separation of nano- and microparticles and large molecular weight solutes such as synthetic polymers and proteins. We report on a theoretical study showing that the separation resolution of open-tubular HDC can be significantly enhanced by changing the cross-sectional shape of the separation channel. By enforcing Brenner's macro-transport approach, we provide theoretical/numerical evidence showing how the shape of the cross section influences quantitatively both the selectivity and the axial dispersion of the suspended particles in HDC. The separation performance of square-, triangle-, and star-shaped channel cross sections is compared to that of a cylindrical capillary over three decades of the particle Péclet number in terms of the minimal separation length and time to obtain the unit resolution of a two-particle mixture. Enhancement factors up to 400% are found in the case of triangular shapes, with the best performing case being the 70.6° angle, which can be obtained by KOH etching of bulk silicon.

The Effect of Molecular Mass of Hydroxyethyl Cellulose on the Performance of Capillary Electrophoretic Separation of Proteins

Capillary electrophoresis (CE) is a versatile analytical separation method in the field of biochemistry. Although it has been proved that the relative molecular mass (Mr) of the polymers determines the threshold concentration of the entangled polymer solution, which will affect the separation performance of DNA molecules, there is still no report on the effect of Mr on the separation performance of proteins. Herein, we have thoroughly performed the CE of proteins ranged from 14.3 kDa to 116 kDa in a mixed hydroxyethyl cellulose (HEC) solution. The mixed solution was obtained with various Mr including 90,000, 250,000, 720,000, and 1,300,000. Then, we found that the mixed polymer provided a high resolution for small protein molecules while increasing the efficiency of large ones. Results demonstrated that the migration time decreased if HEC (1,300,000) was mixed with the lower Mr one, and the mixed solution (1,300,000/250,000) offered the highest resolution. The resolution was negatively correlated with the electric field strength. Finally, we have employed the optimal electrophoretic conditions to separate proteins in human tears, and it showed that lysozyme, lipocalin, and lactoferrin from human tears were successfully resolved in the mixed HEC. Such work indicates that CE has the potential to be developed as a tool for the diagnosis of xerophthalmia, meibomian gland dysfunction, or other eye diseases.

Application of experimental design in HPLC method optimisation for the simultaneous determination of multiple bioactive cannabinoids

The scientific interest in Cannabis sativa L. analysis has been rapidly increasing in recent years, especially for what concerns cannabinoids, plant secondary metabolites which are well known for having many biological properties. High-performance liquid chromatography (HPLC) is frequently used for both the qualitative and quantitative analysis of cannabinoids in plant extracts from C. sativa and its derived products. Many studies have been focused on the main cannabinoids, such as ∆9-tetrahydrocannabinolic acid (∆9-THCA), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA) and their decarboxylated derivatives, such as ∆9-tetrahydrocannabinol (∆9-THC), cannabidiol (CBD) and cannabigerol (CBG). In addition to the abovementioned compounds, the plant produces other metabolites of the same chemical class, and some of them have shown interesting biological activities.In the light of this, it is important to have efficient analytical methods for the simultaneous separation of cannabinoids, which is quite complex since they present similar chemical-physical characteristics. The present work is focused on the use of the Design of Experiments technique (DoE) to develop and optimise an HPLC method for the simultaneous separation of 14 cannabinoids. Experimental design optimisation was applied by using a Central Composite Face-Centered design to achieve the best resolution with minimum experimental trials. Five significant variables affecting the chromatographic separation, including ammonium formate concentration, gradient elution, run time and flow rate, were studied. A multivariate strategy, based on Principal Component Analysis (PCA) and Partial Least Squared (PLS) regression, was used to define the best operative conditions. The developed method allowed for the separation of 12 out of 14 cannabinoids. Due to co-elution phenomena, HPLC coupled with a triple quadrupole mass analyser (HPLC-ESI-MS/MS) was applied, monitoring the specific transitions of each compound in the multiple reaction monitoring (MRM) mode. Finally, the optimised method was applied to C. sativa extracts having a different cannabinoid profile to demonstrate its efficiency to real samples.The methodology applied in this study can be useful for the separation of other cannabinoid mixtures, by means of appropriate optimisation of the experimental conditions.

Absolute Quantification of Pure Free Radical Reagents by Combination of the Effective Magnetic Moment Method and Quantitative Electron Paramagnetic Resonance Method.

An absolute quantitative analysis of free radicals by combining the effective magnetic moment method and the quantitative electron paramagnetic resonance [qEPR] method is proposed. This combined method utilizes the advantages of both the analytical methods and compensates for their disadvantages. In the effective magnetic moment method, the magnetic moment under a constant magnetic field is measured accurately using a superconducting quantum interference device. The qEPR method compares a "primary standard sample" and "secondary standard sample". The effective magnetic moment method was used to determine the purity of the primary standard sample. The qEPR method realizes a simple purity analysis of free-radical reagents with traceability to the International System of Units (SI). The purity of the free radicals by the qEPR method for pure 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl benzoate [4HTB], 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine [TEMPOL], and di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium [DPPH] reagents was obtained with a relative expanded uncertainty of 0.7% for 4HTB to 1.5% for the DPPH. These small uncertainties were almost equal to those of the purity of the primary standard samples and were achieved by adopting in-plane positioning of the measured sample perpendicular to the cylindrical axis of the sample space of the superconducting quantum interference device. Some purity values of the free radicals for these reagents differed from those stated by the manufacturers. This combined method enables short-time quality control of pure radical reagents, instead of quality control by separation analytical methods or titrations.

Propolis: chemical diversity and challenges in quality control.

Propolis is a resinous natural product produced by honeybees using beeswax and plant exudates. The chemical composition of propolis is highly complex, and varies with region and season. This inherent chemical variability presents several challenges to its standardisation and quality control. The present review was aimed at highlighting marker compounds for different types of propolis, produced by the species Apis mellifera, from different geographical origins and that display different biological activities, and to discuss strategies for quality control. Over 800 compounds have been reported in the different propolises such as temperate, tropical, birch, Mediterranean, and Pacific propolis; these mainly include alcohols, acids and their esters, benzofuranes, benzopyranes, chalcones, flavonoids and their esters, glycosides (flavonoid and diterpene), glycerol and its esters, lignans, phenylpropanoids, steroids, terpenes and terpenoids. Among these, flavonoids (> 140), terpenes and terpenoids (> 160) were major components. A broad range of biological activities, such as anti-oxidant, antimicrobial, anti-inflammatory, immunomodulatory, and anticancer activities, have been ascribed to propolis constituents, as well as the potential of these compounds to be biomarkers. Several analytical techniques, including non-separation and separation methods have been described in the literature for the quality control assessment of propolis. Mass spectrometry coupled with separation methods, followed by chemometric analysis of the data, was found to be a valuable tool for the profiling and classification of propolis samples, including (bio)marker identification. Due to the rampant chemotypic variability, a multiple-marker assessment strategy considering geographical and biological activity marker(s) with chemometric analysis may be a promising approach for propolis quality assessment.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11101-022-09816-1.

Analytical Separation Method: A Student’s Perception on Analogy-based Teaching for Terminology

Analytical Separation Method is a chemistry course offered at Universiti Teknologi MARA (UiTM), Malaysia. This course covers the application of chromatographic techniques, whereby students must be able to describe the principles and terminologies used and relate them with suitable analogies. A strong understanding of this course plays a major role in the development of solid knowledge amongst graduates to survive in the job market. An auspicious method to understand the analytical separation method has not been identified so far, therefore, limits their opportunities for career development in the future. According to the Pedagogical Content Knowledge (PCK) method, teachers must first understand the knowledge before presenting the information to students during the teaching process. If we can recognise this concept clearly, efforts towards the teaching of science will lead to a new achievement. Analogy-based teaching was found to be capable of increasing students' understanding with regards to the subject matter. The present research successfully identified the level of understanding towards terminology and analogy along with the readiness of students to use an alternative teaching method across campuses, and the readiness concerning the usage of different interactive teaching tools. Quantitative measurement was conducted via SPSS using a sample of 128 students from four different campuses. Based on the analysis, most of the students faced difficulties in accurately translating and relating to the terminologies (57.8%) and analogies (55.5%) concerning the theories without proper guidance. According to the overall mean score (4.18), the range of positive agreement signified the student’s readiness towards the usage of interactive tools during teaching and learning sessions. There was no significant difference in students’ level of understanding on analogy across campuses, however, a significant difference was found in students’ level of understanding on terminologies used across campuses. In pairwise comparisons, there was a statistically significant difference (p-value = 0.009 &lt; 0.05) between Kuala Pilah (K) and Arau (A) campuses.

Simultaneous determination of actinides and 90Sr in large-size soil and sediment samples

A simultaneous analytical method for sequential separation and determination of actinides and 90Sr in large-size environmental samples has been developed. In this method, successive co-precipitation steps were firstly conducted to remove matrix elements, then sequential column separation method was applied for simultaneous separation and purification of actinides and 90Sr/90Y. By using vacuum box technology, the total analytical time was minimized and batch processing allowed analyzing 12 samples in four days. The activity of 90Sr was obtained immediately by measuring its daughter radionuclide (90Y) with triple-to-double coincidence ratio (TDCR) Cherenkov counting, while the concentrations of Pu isotopes and 241Am could be measured by alpha spectrometry and mass spectrometric techniques. The overall recoveries of Pu, Am, Sr and Y were higher than 70% for the entire procedure, while the recovery ratios of Sr/Y were between 0.95 and 1.04 before chromatographic separation. The developed method was verified using 20 g and 50 g of environmental soil samples spiked with certified reference materials IAEA-384 or IAEA-385 and standard solution of 90Sr/90Y, and good agreement between the expected values and measured results has been achieved.

Single sample preparation for the simultaneous extraction of drugs, pharmaceuticals, cannabinoids and endogenous steroids in hair.

Recently, we published a multi-analyte method for the simultaneous analysis of 116 drugs and pharmaceuticals including different substance groups like opioids, stimulants, benzodiazepines, z-drugs, antidepressants and neuroleptics based on a single sample workup followed by a single analytical measurement with LC-MS/MS. However, in some cases, additional analysis of further substance groups, such as cannabinoids and endogenous steroids, is required, which are analyzed in our laboratory using separate sample preparation and separate analytical methods. The goal of this study was to use the knowledge from the different sample preparations and combine them into a single sample preparation and extraction workflow for the simultaneous extraction of drugs, pharmaceuticals, cannabinoids, and endogenous steroids to be analyzed with the appropriate analytical methods. A partial validation of selected parameters such as selectivity, linearity, limit of quantification (LOQ), accuracy, precision and robustness for the different analytical methods was carried out and revalidated. In addition, comparative measurements of quality controls and authentic pools were performed and statistically evaluated using the unpaired t-test or the non-parametric Mann-Whitney test. The results using the newly established sample preparation and extraction were in good agreement with the original data. In conclusion, the newly established sample preparation is suitable for the combined extraction of drugs, pharmaceuticals, cannabinoids and endogenous steroids, and gives reliable results for quantification of various substances.