Journal of Chromatography B (v.934, #C)

A liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed and validated for a PEGylated adnectin therapeutic protein in cynomolgus monkey plasma. The validated method was performed using protein precipitation coupled with trypsin digestion, followed by LC–MS/MS detection of a surrogate peptide generated from the PEGylated adnectin protein. A tryptic peptide generated from a PEGylated adnectin protein analog was used as the internal standard to standardize the digestion, extraction, and quantitation processes. The protein precipitation extraction of the protein from cynomolgus plasma was performed using an acidic 2-propanol organic solution. Following the extraction, the supernatant was removed and a 45 min trypsin digestion was performed at 60 °C on the supernatant layer. The linear dynamic range of the assay was 50.0–25,000 ng/mL. Chromatographic separation was performed with an Acquity BEH C18 (1.7 μm particle size, 2.1 mm × 50 mm) column using gradient elution. The assay proved to have robust accuracy, precision, and stability for the representative surrogate peptide of the PEGylated adnectin protein being evaluated. The validated method was implemented as a high throughput assay for a PEGylated adnectin protein using a similar PEGylated adnectin therapeutic protein as the internal standard that can be used for future monkey toxicokinetic (TK) studies
Keywords: LC–MS/MS; Bioanalytical; Method validation; PEGylated adnectin; Peptide; Trypsin digestion;

Ultra-performance liquid chromatography–tandem mass spectrometry determination and depletion profile of flunixin residues in tissues after single oral administration in rabbits by Ai-Ling Zhu; Tao Peng; Liang Liu; Xi Xia; Ting Hu; Xiao-Qi Tao; Kai Wen; Lin-Li Cheng; Jian-Cheng Li; Shuang-Yang Ding; Xing-Yuan Cao; Hai-Yang Jiang (8-15).
An ultra-performance liquid chromatography with tandem mass spectrometric detection (UPLC–MS/MS) method was developed for the detection of flunixin residues in rabbit tissues. The samples were extracted with acidic acetonitrile, defatted with n-hexane, and then purified by HLB solid-phase extraction cartridge. Analysis was carried out on UPLC–ESI-MS/MS working with multiple reaction monitoring (MRM) mode. The limits of detection (LODs) of the method were 0.3–0.8 μg kg−1 and limits of quantification (LOQs) were 1.0–3.0 μg kg−1 in rabbit tissues, respectively. In all fortified samples at a concentration range of 1.0–300.0 μg kg−1, mean recoveries were 61.7–115.7% with relative standard deviations (RSDs) below 16%. Residue depletion of flunixin in rabbit was conducted after oral administration at a dose of 5 mg kg−1 of body weight. The average concentrations for flunixin measured 2 h post-administration in kidney and intestine were significantly higher than in liver, heart and muscle. The concentrations for flunixin in all rabbit tissues were below the LOD or not detected in all tissues after 96 h administration of drug. A minimum withdrawal time of 21 h was indicated for residue levels in heart, liver, kidney, intestine and muscle below the maximum residue limits (MRLs).
Keywords: Ultra-performance liquid chromatography tandem mass spectrometry; Flunixin; Rabbit tissue; Residue depletion; Withdrawal time;

Multiplexed analysis of steroid hormones in human serum using novel microflow tile technology and LC–MS/MS by Carolyn J. Broccardo; Kevin L. Schauer; Wendy M. Kohrt; Robert S. Schwartz; James P. Murphy; Jessica E. Prenni (16-21).
A novel microfluidic chromatography device coupled with tandem mass spectrometry (LC–MS/MS) was utilized for the multiplex analysis of 5 steroids (testosterone, dihydrotestosterone, progesterone, cortisol, cortisone) in human serum. The use of microfluidics allowed for reduction of the chromatographic flow rate to 3 μl/min with overall method run times comparable to standard flow LC–MS/MS methods reported in the literature, corresponding to a 150 fold decrease in solvent consumption. Furthermore, a simple sample preparation protocol was employed requiring injection of only 0.5 μl of sample, corresponding to a 100400 fold increase in on-column sensitivity as compared to published standard flow assays. The measured LOQ for both testosterone and progesterone was 0.4 ng/mL, representing an improvement over reported literature values obtained by standard flow methods employing comparable sample preparation and large injection volumes. The LOQs for cortisol (1.9 ng/mL), cortisone (0.3 ng/mL), and dihydrotestosterone (1.4 ng/mL) were all within a biologically relevant range. A comparison of clinical serum samples was performed for the analysis of testosterone using this microfluidic LC–MS/MS assay and the Beckman Access II automated antibody-based measurement system. The immunoassay results were systematically higher due to matrix interference which was easily resolved with the increased chromatographic resolution obtained in the microflow LC–MS/MS assay.
Keywords: Steroid; MRM; Microflow; Serum; Immunoassay; LC–MS/MS;

Liquid chromatography–tandem mass spectrometry assay for the EGFR inhibitor pelitinib in plasma by Dino Luethi; Selvi Durmus; Alfred H. Schinkel; Jan H.M. Schellens; Jos H. Beijnen; Rolf W. Sparidans (22-25).
A bioanalytical liquid chromatography–tandem mass spectrometry assay for the tyrosine kinase inhibitor pelitinib was developed and validated in plasma. Acetonitrile containing the internal standard erlotinib was used to precipitate proteins. The extract was diluted with water and then directly injected onto the sub-2 μm particle, bridged ethylsilica hybrid trifunctional bonded C18 column. A gradient consisting of 0.02% (v/v) formic acid in a methanol–water mixture was used. The ionization mode of the electrospray interface was positive and the analyte was detected by a triple quadrupole mass spectrometer in the selected reaction monitoring mode. The calibration range of the assay was 1–200 ng/ml. The within day precision, the between day precision, and the accuracy were 3.5–7.4%, 4.5–8.6%, and 94.0–104.8%, respectively. The stability of the drug was sufficient under all relevant conditions. The validated assay was used to measure drug levels in wild-type FVB mice and pharmacokinetic parameters were assessed.
Keywords: Pelitinib; LC–MS/MS; EGFR inhibitor; Bioanalytical assay; FVB mouse;

Immobilized metal affinity chromatography and human serum proteomics by Fengrong Wang; Christyne Chmil; Frank Pierce; Kulothungan Ganapathy; Brooks B. Gump; James A. MacKenzie; Yehia Mechref; Kestutis Bendinskas (26-33).
Metal-binding proteins have a pivotal role in normal and diseased states. We used metal affinity chromatography to enrich a fraction of human serum proteins on immobilized columns loaded with cadmium, nickel, zinc, copper, or lead in bis–Tris saline and these proteins were identified using LC–MS/MS. Tens of enriched proteins were identified and we here present the 20 most abundant for binding each metal. The binding of various proteins (complement C3, alpha-2-macroglobulin, serum albumin, apolipoprotein B-100, complement component 4B preproprotein, apolipoprotein A–I, serotransferrin, alpha-1-antitrypsin, ceruloplasmin, 47 kDa protein, uncharacterized protein DKFZp686P15220, transthyretin, hemopexin, inter-alpha-trypsin inhibitor heavy chain H2, and histidine-rich glycoprotein) to different metals using immobilized metal affinity chromatography was compared to the literature. Although many metal-binding properties of these proteins have been confirmed, new metal-binding proteins have also been identified. The metal array use in the proteomic biomarker search technologies gives this data particular importance.
Keywords: Immobilized metal affinity chromatography; Human serum; Proteomics; LC–MS/MS; ICP-MS; Metal binding proteins;

Cefpodoxime proxetil (CFP), a broad-spectrum third-generation cephalosporin, has been used most widely in the treatment of respiratory and urinary tract infections. For bioequivalence study of CFP in rabbit plasma, it was necessary to develop a highly sensitive and selective high-performance liquid chromatographic (HPLC) method with fluorescence (FL) detection. The pre-column labeling of cefpodoxime acid (CFA) (active metabolite) with an efficient benzofurazan type fluorogenic reagent, 4-N,N-dimethyl aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was carried out in the present study in 100 mM borate buffer (pH = 8.5) at 50 °C for 15 min. The obtained fluorescent products were separated on C18 column with an isocratic elution of the mobile phase, which consists of 10 mM phosphate buffer (pH = 3.5)/CH3CN (70:30, v/v). The fluorescent product (DBD-CFA) was detected fluorimetrically at 556 nm with an excitation wavelength of 430 nm. Cefotaxime sodium was used as internal standard. The method was validated according to the requirements of US-FDA guidelines. The correlation coefficient of 0.999 was obtained in the concentration ranges of 10–1000 ng mL−1. The limits of detection and quantification (S/N = 3) were 3 and 10 ng mL−1, respectively. Plasma CFA levels were successfully determined in rabbit with satisfactory precision and accuracy. The proposed HPLC-FL method was successfully applied to study bioequivalence in rabbits for two formulations of different brands contained CFP (prodrug) in a randomized, two-way, single-dose, crossover study and all pharmacokinetic parameters for the two formulations were assessed.
Keywords: Cefpodoxime acid; Prodrug; Benzofurazan; Bioequivalence study; Pharmacokinetic;

Simultaneous determination of nicotine and cotinine in serum using high-performance liquid chromatography with fluorometric detection and postcolumn UV-photoirradiation system by Makoto Yasuda; Tatsuhiro Ota; Atsushi Morikawa; Ken-ichi Mawatari; Tomoko Fukuuchi; Noriko Yamaoka; Kiyoko Kaneko; Kazuya Nakagomi (41-45).
A simple and rapid method for the simultaneous determination of serum nicotine and cotinine using high-performance liquid chromatography (HPLC)-fluorometric detection with a postcolumn ultraviolet-photoirradiation system was developed. Analytes were extracted from alkalinized human serum via liquid–liquid extraction using chloroform. The organic phase was back-extracted with the acidified aqueous phase, and the analytes were directly injected into an ion-pair reversed-phase HPLC system. 6-Aminoquinoline was used as an internal standard. Nicotine, cotinine, and 6-aminoquinoline were separated within 14 min. The extraction efficiency of nicotine and cotinine was greater than 91%. The linear range was 0.30–1000 ng for nicotine and 0.06–1000 ng for cotinine. In serum samples from smokers, the concentrations of nicotine and cotinine were 8–15 ng/mL and 156–372 ng/mL, respectively.
Keywords: Nicotine; Cotinine; UV-photoirradiation; Liquid–liquid extraction; HPLC; Fluorescence detection;

Separation and purification of hyaluronic acid by embedded glucuronic acid imprinted polymers into cryogel by Özlem Biçen Ünlüer; Arzu Ersöz; Adil Denizli; Rasime Demirel; Rıdvan Say (46-52).
Hyaluronic acid (HA) has been used in many applications such as pharmaceutical, clinical and cosmetics, so its separation and purification is very important. In this study, firstly d-glucuronic acid imprinted polymers (MIPs) have been synthesized for the separation of HA which has glucuronic acid part in its structure. MIP particles have characterized by elemental analysis, Fourier Transform Infrared Spectroscopy (FT-IR) and swelling tests. Then, synthesized MIP particles have embedded into polyacrylamide based cryogel. Cryogel has prepared by free radical cryogelation process initiated by N,N,N′,N′-Tetramethylethylenediamine (TEMED) and ammonium persulfate (APS) as redox initiators. This cryogel material was characterized by FT-IR, swelling tests, scanning electron microscopy (SEM) and surface adsorption analyze including pore size analyzer (BET) method. The adsorption of HA has investigated by spectrophotometric method using MIPs embedded into cryogel columns (GAIPEC) and the maximum HA adsorption capacity was found to be 318 mgg−1. The selectivity of GAIPEC column has estimated using N-acetylglucose amine as interfering agent since this molecule is a part of HA and the results have shown that GAIPEC has been nearly 35 times selective for HA than N-acetylglucose amine. The optimum chromatographic conditions for separation of HA were investigated. pH 7.0 buffer solution for elution and 0.1 M of NaCl solution as desorption agent were used at 0.5 mL min−1 flow rate. Also, recovery of GAIPEC was investigated and the results have shown that GAIPEC could be used many times without decreasing its adsorption capacity significantly. Here in, combining selectivity of MIP particles and mechanical properties of cryogel, a rigid and stable material was prepared for the separation and purification of HA. To point out this, HA has been isolated from fish eye and fermentation of Streptococcus equi RSKK 679 cell culture. After that, it has characterized and Fast Protein Liquid Chromatography (FPLC) applications have been investigated
Keywords: Cryogel; d-Glucuronic acid; FPLC; Hyaluronic acid; Molecularly imprinted polymers;

Ethylenethiourea (ETU) is of major toxicological concern, since in experimental animal studies, ETU has shown a large spectrum of adverse effects. High occupational exposure can be found among agricultural workers or during manufacturing of ethylenbisdithiocarbamates (EBDC). For the general public, sources of environmental exposure may be residues of ETU in commercial products, food and beverages. For the determination of ETU in human urine we present a high-throughput online on-column extraction liquid chromatography triple quadrupole mass spectrometry method using direct injection of hydrolysed urine samples. This method is simple, user- and environmentally friendly and all sample preparation is performed in 96-well plates. A labelled ETU internal standard was used for quantification. The method showed a good sensitivity with a limit of quantification (LOQ) of 0.5 ng ETU/mL urine and the calibration curve was linear in the range 0.25–200 ng ETU/mL urine. The within-run, between-run and between-batch precision was between 6% and 13%. Alkaline hydrolysis considerably increased the levels of ETU indicating a potential conjugate. The method was applied in an experimental dermal exposure study in humans, with sample concentrations ranging from 0.4 to 5.0 ng ETU/mL urine. The excretion in urine was 10% of the applied dose. The elimination profile seemed to differ between the two individuals. The results show an estimated half-life of ETU between 34 and 72 h. Although the experiment is limited to two individuals, the data provide valuable and new information regarding the toxicokinetics of ETU after dermal exposure.
Keywords: Biomarkers; Dermal; Ethylenethiourea; High-throughput; LC/MS/MS; Urine;

Determination of exposure to cyanide can be accomplished by direct cyanide analysis or indirectly by analysis of cyanide detoxification products, such as thiocyanate and 2-amino-2-thiazoline-4-carboxylic acid. A potentially important marker and detoxification product of cyanide exposure, α-ketoglutarate cyanohydrin (α-KgCN), is produced by the reaction of cyanide and α-ketoglutarate. Therefore, an ultra high-performance liquid chromatography tandem mass spectrometry method to determine α-KgCN in plasma was developed. Swine plasma was spiked with α-KgCN and α-KgCN-d4 (internal standard) and proteins were precipitated with 1% formic acid in acetonitrile. After centrifugation, the supernatant was dried, reconstituted, separated by reversed phase high performance liquid chromatography and analyzed by tandem mass spectrometry. The method produced a dynamic range of 0.3–50 μM and a detection limit of 200 nM for α-KgCN. Furthermore, the method produced a %RSD of less than 13% for all intra- and inter-assay analyses. The stability of α-KgCN was poor for most storage conditions tested, except for −80 °C, which produced stable concentrations of α-KgCN for the 30 days tested. The validated method was tested by analysis of α-KgCN in the plasma of cyanide-exposed swine. α-KgCN was not detected pre-exposure, but was detected in all post-exposure plasma samples tested. To our knowledge, this method is the first reported analytical method for detecting α-KgCN in any matrix.
Keywords: α-Ketoglutarate cyanohydrin; Liquid chromatography tandem mass-spectrometry; Cyanide; Method development; Biomarkers;

Colorful quality control of chromatographic sample preparation by J. Pesek; Th. Krüger; B. Tautkus; H. Rhode (66-70).
Multidimensional chromatographic separation for proteomic biomarker search generates sets of several hundred homologous fractions, which have to be compared. Due to the high number of sequential steps, deviations between samples may be produced randomly by slight processing differences. These deviations may falsify proteomic results. In order to overcome this problem, we tested the applicability of quality control by colored phycobilins as internal standards. The elution of the used protein standards themselves shows a high reproducibility since their main peak location is practically constant under proper performance of size exclusion and anion exchange chromatography. This applies to runs of one phycobilin alone, combined with another phycobilin, or combined with plasma proteins. Thus, these protein standards do not disturb sample processing. Characteristic peak shifts of phycobilins allow easy observation of deviations caused by typical failures in the elution protocol (aberrant step number, buffer permutation). Mass spectrometric analysis is not influenced by their presence since protein coverage, peptide numbers, and protein numbers are not altered. Thus, colored protein standards may be used for quality control and evaluation of robustness of various chromatographic applications
Keywords: Chromatographic sample preparation; Quality control; Phycobilins;

Interfacial partitioning behaviour of bovine serum albumin in polymer-salt aqueous two-phase system by Yin Hui Chow; Yee Jiun Yap; Mohd. Shamsul Anuar; Bimo Ario Tejo; Arbakariya Ariff; Pau Loke Show; Eng-Poh Ng; Tau Chuan Ling (71-78).
A relationship is proposed for the interfacial partitioning of protein in poly(ethylene glycol) (PEG)-phosphate aqueous two-phase system (ATPS). The relationship relates the natural logarithm of interfacial partition coefficient, ln G to the PEG concentration difference between the top and bottom phases, Δ[PEG], with the equation ln G = AΔ[PEG] +  B. Results showed that this relationship provides good fits to the partition of bovine serum albumin (BSA) in ATPS which is comprised of phosphate and PEG of four different molecular weight 1450 g/mol, 2000 g/mol, 3350 g/mol and 4000 g/mol, with the tie-line length (TLL) in the range of 44–60% (w/w) at pH 7.0. The decrease of A values with the increase of PEG molecular weight indicates that the correlation between ln G and Δ[PEG] decreases with the increase in PEG molecular weight and the presence of protein–polymer hydrophobic interaction. When temperature was increased, a non-linear relationship of ln G inversely proportional to temperature was observed. The amount of proteins adsorbed at the interface increased proportionally with the amount of BSA loaded whereas the partition coefficient, K remained relatively constant. The relationship proposed could be applied to elucidate interfacial partitioning behaviour of other biomolecules in polymer-salt ATPS.
Keywords: Aqueous two-phase system; Interfacial partition; Statistical mechanics; Bioseparation; Protein recovery; Purification;

Analysis of chloroformate-derivatised amino acids, dipeptides and polyamines by LC–MS/MS by Baljit K. Ubhi; Peter W. Davenport; Martin Welch; John Riley; Julian L. Griffin; Susan C. Connor (79-88).
A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed which, with sample preparation using a commercially available kit, allows rapid quantitation of 39 chloroformate-derivatised amino acids (AAs), polyamines (PAs) and dipeptides (DPs) in complex biological matrices. Lower limits of quantitation (LOQ) were 20–150 nM for putrescine, spermine, spermidine, cadaverine, agmatine, and below 5 μM for all analytes. Responses were linear for all analytes between 0.5 and 50 μM. Quantitative measurements of all 39 metabolites were achieved within a 15 min runtime. The method was evaluated with a Pseudomonas aeruginosa cell extract study (n  = 24) and a larger human urine study (n  = 308). Batch effects were observed in the urine study and an investigation of instrument and sample stability showed a wave-like pattern in the MS responses. Both the run order and inter-batch variation were successfully corrected by normalising to pooled urine quality control data. Thus, this method should be suitable for diverse biological matrices and for large as well as small sample sets.
Keywords: LC–MS/MS; Amino acids; Metabolites; Metabolomics;

A highly selective and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous determination of trantinterol (SPFF) and its major metabolites for the first time. The analytes were extracted from rat urine and feces samples by liquid–liquid extraction (LLE) and determined in multiple reaction monitoring (MRM) mode with clenbuterol as the internal standard. Chromatographic separation was achieved on a Venusil ASB C8 column (2.1 mm × 100 mm, 3 μm), with the mobile phase consisted of methanol–0.2% formic acid (30:70, v/v) at the flow rate of 0.2 mL/min. Each sample was chromatographed within 5 min. This method has a lower limit of quantification (LLOQ) of 0.450, 1.05, 1.35, 0.904 and 1.36 ng/mL for trantinterol (SPFF), arylhydroxylamine trantinterol (N-OH-SPFF), tert-butyl hydroxylated trantinterol (Tert-OH-SPFF), 1-carbonyl trantinterol (SPFF-COOH) and 3-methyl sulfone-dechloro-trantinterol (SPFF-SO2CH3) in rat urine, and 0.450, 1.35 and 0.904 ng/mL for SPFF, Tert-OH-SPFF and SPFF-COOH in rat feces, respectively. The linear correlation coefficients were greater than 0.990. The intra- and inter-day precision (relative standard deviation, RSD) values were below 15% and the accuracy (relative error, RE) was −9.9% to 11% at three quality control levels. The method has been successfully applied to the excretion study following an oral administration of 1 mg/kg trantinterol to rats.
Keywords: Trantinterol; Metabolites; LC–MS/MS; Urine; Feces; Excretion;

In this study, we were able to develop a simple analytical procedure to assay the presence of two antimicrobial agents, triclosan (TCS) and triclocarban (TCC), in human nails. Samples were digested using sodium hydroxide (NaOH), extracted using dichloromethane, and analyzed using ultra performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry operating in the negative ion mode. Mean recoveries were performed at three fortification levels ranging from 98.1% to 106.3% with relative standard deviations between 1.8% and 18.1% (n  = 6). The limits of quantification (LOQ) for the method were 2.0 and 0.2 μg/kg for TCS and TCC, respectively. Both compounds were ubiquitously found in all real samples (n  = 20) with concentrations ranging from μg/kg to several mg/kg.
Keywords: Triclosan; Triclocarban; Nail; Liquid–liquid extraction; LC–MS/MS;

Detection and tentative identification of urinary phase I metabolites of phenylacetylindole cannabimimetics JWH-203 and JWH-251, by GC–MS and LC–MS/MS by Pierce Kavanagh; Andrej Grigoryev; Alexandra Melnik; Sergey Savchuk; Anton Simonov; Vladimir Rozhanets (102-108).
The synthetic phenylacetylindole cannabimimetics, JWH-203 and JWH-251, have been identified in ‘herbal’ smoking mixtures following the widespread legislative control of ‘first generation’ compounds such as JWH-018 and CP47, 497(C8). N-Alkylindole cannabimimetics (including phenylacetylindoles) undergo extensive metabolism and little or none of the parent compounds are found in urine. Utilizing GC–MS and LC–MS/MS, a series of JWH-203 and JWH-251 urinary metabolites have been tentatively identified. These are products of mono- and dihydroxylation, monohydroxylation combined with formation of carbonyl group on the N-pentyl chain, carboxylation of N-pentyl chain and N-dealkylation combined with monohydroxylation. Additionally, trihydroxylated metabolites were detected for JWH-203. No parent compounds were detected. The monohydroxylated metabolites with the hydroxyl group positioned on the N-pentyl chain were the most abundant and were found to be suitable for establishing ingestion of JWH-203 or JWH-250. Maximum urinary concentrations of chain-monohydroxylated metabolites were observed at 2.5–3 h (JWH-203) and 6–10 h (JWH-251) following ingestion. These metabolites were observed (GC–MS) for to 10 and 8 days (JWH-203 and JWH-251, respectively).
Keywords: Synthetic cannabinoid; JWH-203; JWH-251; Metabolite; GC–MS; LC–MS/MS;

Preparation of metallic pivot-based imprinted monolith for polar template by Dan-Dan Zhong; Yan-Ping Huang; Xue-Lei Xin; Zhao-Sheng Liu; Haji Akber Aisa (109-116).
One of the main challenges in MIPs preparation is the proper MIP monolith design for water-soluble compounds due to the difficulty in satisfying the demands of both good column permeability and affinity to polar template. A new strategy of metallic pivot in a ternary porogenic system of dimethyl sulfoxide (DMSO)-dimethylformamide (DMF)-1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF4) was suggested to solve this problem. An imprinted monolithic column with high porosity and good permeability was synthesized using a mixture of methyl gallate (template), 4-vinylpyridine, ethylene glycol dimethacrylate, and cobalt acetate. Some polymerization factors, such as template-monomer molar ratio and the composition of the ionic liquid, on the imprinting effect of the resulting MIPs monoliths were systematically investigated. In a mobile phase of acetonitrile-buffer, the greatest imprinting factor of 10.9 was obtained on the MIPs monolith with the optimized polymerization parameters. Thermodynamic analysis for separation demonstrated that the separation between the template and its analogs on the ion-mediated MIPs monolith is an enthalpy-controlled process.
Keywords: Monolith; Molecularly imprinted polymer; Metal ions; Molecular recognition; Methyl gallate; Ionic liquid;

Linear calibration is usually performed using eight to ten calibration concentration levels in regulated LC–MS bioanalysis because a minimum of six are specified in regulatory guidelines. However, we have previously reported that two-concentration linear calibration is as reliable as or even better than using multiple concentrations. The purpose of this research is to compare two-concentration with multiple-concentration linear calibration through retrospective data analysis of multiple bioanalytical projects that were conducted in an independent regulated bioanalytical laboratory. A total of 12 bioanalytical projects were randomly selected: two validations and two studies for each of the three most commonly used types of sample extraction methods (protein precipitation, liquid–liquid extraction, solid-phase extraction). When the existing data were retrospectively linearly regressed using only the lowest and the highest concentration levels, no extra batch failure/QC rejection was observed and the differences in accuracy and precision between the original multi-concentration regression and the new two-concentration linear regression are negligible. Specifically, the differences in overall mean apparent bias (square root of mean individual bias squares) are within the ranges of −0.3% to 0.7% and 0.1–0.7% for the validations and studies, respectively. The differences in mean QC concentrations are within the ranges of −0.6% to 1.8% and −0.8% to 2.5% for the validations and studies, respectively. The differences in %CV are within the ranges of −0.7% to 0.9% and −0.3% to 0.6% for the validations and studies, respectively. The average differences in study sample concentrations are within the range of −0.8% to 2.3%. With two-concentration linear regression, an average of 13% of time and cost could have been saved for each batch together with 53% of saving in the lead-in for each project (the preparation of working standard solutions, spiking, and aliquoting). Furthermore, examples are given as how to evaluate the linearity over the entire concentration range when only two concentration levels are used for linear regression. To conclude, two-concentration linear regression is accurate and robust enough for routine use in regulated LC–MS bioanalysis and it significantly saves time and cost as well.
Keywords: Linear regression; Two-point calibration; Regulated bioanalysis; LC–MS; Retrospective analysis;