A review of analytical parameters in 'rapid' liquid chromatographic methods for bioanalysis: Can we do better?

Rapid bioanalysis is beneficial to many applications. However, how 'rapid' a method is, or could be, is often an unanswered question. In this statistical review, the authors have assessed multiple pre-analytical (i.e. sample preparation), and analytical method parameters specifically for liquid chromatography to assist researchers in developing and validating 'rapid' bioanalytical methods. We restricted the search to urine and plasma matrices only. Data were extracted from over 2,000 recent studies and evaluated to assess how these parameters affected the 'on-instrument' analysis time. In addition to methods using ultra-violet (UV) detection, there were a large number of mass spectrometric (MS) methods, allowing additional review of the differences between high- and low-resolution MS on analysis time. We observed that most (N = 922, 70 %) methods used 5 or 10 cm columns, and that whilst uptake of ultra-high performance (U)HPLC columns was good, the use of sub-5 cm columns and/or flow rates in excess of 1 mL/min was incredibly rare (N = 25, 3 %). The detector of choice for quantitative (U)HPLC-MS remains the triple quadrupole, although a number of groups report the use of high-resolution MS for such methods.

Post mortem blood bromazolam concentrations and co-findings in 96 coronial cases within England and Wales.

Bromazolam is a newly emerging benzodiazepine drug which is not licensed for medicinal use. It may be sourced as a New Psychoactive Substance (NPS) for its desired effects or be consumed unknowingly via counterfeit Xanax® or Valium® preparations. As part of our Coronial workload, we observed an increase in the detection of bromazolam from September 2021 to November 2022. We report a series of 96 cases in which bromazolam was quantitated by high resolution accurate mass - mass spectrometry (HRAM - MS) in post-mortem blood. The mean (SD) post-mortem blood bromazolam concentration from our case series was 64.6 ( ± 79.4) µg/L (range <1-425 µg/L). Routine toxicological screening results have also been reported; the most commonly encountered drugs taken in combination with bromazolam were cocaine, gabapentinoids and diazepam. In 48% of cases at least one further designer benzodiazepine drug was also present (etizolam, flualprazolam, flubromazolam, flubromazepam). It is essential that laboratories providing toxicological investigations are aware of the limitations of their assays; and inclusion of bromazolam within targeted screening panels using LC-MS/MS is encouraged. Bromazolam has not been associated with death in isolation from resulting toxic concentrations; however, it is likely to enhance adverse clinical effects when taken in combination with stimulant and/or centrally-acting depressant drugs (poly-drug deaths). Bromazolam, similar to other benzodiazepines, may also impair cognition and decision making skills.

Plasma and Urine Levamisole in Clinical Samples Containing Benzoylecgonine: Absence of Aminorex.

Aminorex has been reported as a metabolite of levamisole in man, but data on the aminorex concentrations in clinical samples are scant. We thus measured levamisole, aminorex and benzoylecgonine in urine, and levamisole and aminorex in plasma using achiral liquid chromatography-high resolution mass spectrometry. Centrifuged urine (50 µL) was diluted with LC eluent containing internal standard (benzoylecgonine-D3, 25 µg/L) (450 µL). For plasma, sample (200 µL) and Tris solution (2 mol/L, pH 10.6, 100 µL) were added to a 60.5 × 7.5 mm i.d. glass test tube. Internal standard solution (ketamine-D4, 200 µg/L) (10 µL) was added and the tube contents vortex-mixed (5 s). Butyl acetate:butanol (9 + 1, v/v; 200 µL) was added and after vortex-mixing (30 s) and centrifugation (13,680 × g, 4 min), the extract was evaporated to dryness and reconstituted in 10 mmol/L aqueous ammonium formate containing 0.1% (v/v) formic acid (150 µL). Prepared samples and extracts (100 µL) were analyzed using an AccucoreTM Phenyl-Hexyl column (2.6 mm a.p.s., 100 × 2.1 mm i.d.) maintained at 40°C. MS detection was in positive mode using heated electrospray ionization (ThermoFisher Q-ExactiveTM). Intra- and inter-assay accuracy and precision were ±20%, and ≤11%, respectively, for all analytes in both matrices. Lower limits of quantitation were 0.1 and 1 µg/L (all analytes) in plasma and urine, respectively. Of 100 consecutive urine samples submitted for drugs of abuse screening containing benzoylecgonine, levamisole was detected in 72 (median 565, range 4-72,970 µg/L). Levamisole was also measured in eight plasma samples (median 10.6, range 0.9-64.1 µg/L). A number of metabolites of levamisole (4-hydroxylevamisole, levamisole sulfoxide, levamisole glucuronide, and hydroxylevamisole glucuronide) were tentatively identified in urine. Neither aminorex, nor any of its reported metabolites were detected in any sample.

The use of turbulent flow chromatography for rapid, on-line analysis of tryptic digests.

RATIONALE: Following digestion of proteins with trypsin, digests are typically subjected to further 'clean-up' prior to liquid chromatography/mass spectometry (LC/MS) analysis, in order to reduce the complexity of the digested matrix, as well as helping to remove residual denaturants and reduction/alkylation reagents prior to injection onto the analytical HPLC column. Often, this is carried out using off-line techniques, and is not ideally suited to high-throughput workloads, for example in clinical laboratories. METHODS: Bovine serum albumin (BSA) was used as a model protein. Following denaturation with urea, reduction/alkylation, and digestion with trypsin, the analytical recovery of a selection of proteotypic BSA peptides was assessed using a two-dimensional, turbulent flow chromatography method. Peptides were identified using a Q Exactive™ mass spectometer operating in full-scan mode. RESULTS: Total analysis time (including the on-line sample clean-up) was 15 min per injection. Aside from the most hydrophilic peptide selected, ATEEQLK, recovery using the turbulent flow chromatography systems was greater than 30% for all remaining peptides (N = 17), and exceeded 50% for 12 of the 18 peptides studied. There was a broad correlation between the hydrophobicity factor and the observed recovery. CONCLUSIONS: This study suggests that turbulent flow chromatography offers a rapid, on-line alternative to solid-phase extraction for the analysis of peptide digests by LC/MS. A wide range of column chemistries are available, and the technique can be further optimised for analyses which are targetted to specific peptides. As with turbulent flow chromatography for small-molecule workflows, this approach may be ideally suited to high-throughput applications, such as those which are emerging from within clinical laboratories.

Commercial insulin immunoassays fail to detect commonly prescribed insulin analogues.

OBJECTIVES: Blood insulin and C-peptide are key investigations in the differential diagnosis of hypoglycaemia. Analogues of insulin have modified primary-sequences compared to native human insulin, as such may not cross react with insulin assays. This has important implications in detecting surreptitious or malicious insulin administration. The aim of this study is to assess the cross-reactivity of all insulins currently listed in the British National Formulary (BNF65, 2013) in clinical insulin assays currently used in UK clinical laboratories. DESIGN AND METHODS: Sample sets were prepared for all 15 exogenous insulin classes listed in the BNF, at concentrations of 1000 pmol/L and 300 pmol/L, using pooled human serum. Samples were sent blinded to 5 participating analytical laboratories to cover analysis on the 10 major clinical insulin assays used in the UK. RESULTS: The ability of insulin assays to detect exogenous insulin preparations was highly variable and ranged from 0% to >140% for a single exogenous insulin. Four assays were highly specific for the human insulin sequence and had no cross-reactivity with any synthetic analogue insulin. Two detected all insulin types (human sequence, animal and synthetic analogue), with the remaining having variable cross-reactivity. CONCLUSION: The cross-reactivity of the 15 exogenous insulin preparations is highly variable in the assays used in clinical laboratories around the UK. It is important that laboratories and clinicians are aware of the limitations of their local assays to avoid missing the important diagnosis of hypoglycaemia secondary to excessive exogenous insulin. Where necessary, samples should be referred to specialist centres for insulin analysis and ideally by a validated and fully-quantitative mass spectrometry-based method.

False-positive buprenorphine by CEDIA in patients prescribed amisulpride or sulpiride.

Buprenorphine is a potent partial opioid agonist that is analyzed in urine to (i) monitor adherence to maintenance or detoxification therapy and (ii) detect illicit use. Buprenorphine analysis is commonly conducted on urine by immunoassay, but is subject to cross-reactivity from other drugs/drug metabolites, including morphine, codeine and dihydrocodeine. This study reports false-positive buprenorphine analysis [Thermo Fisher Scientific cloned enzyme donor immunoassay (CEDIA)] in patients who denied unauthorized buprenorphine use prior to sampling, but who had been prescribed amisulpride. In two cases, confirmatory analysis by liquid chromatography-tandem mass spectrometry was negative (<0.5 µg/L) for buprenorphine and metabolites and positive for amisulpride. Although the cross-reactivity of amisulpride and sulpiride in the CEDIA buprenorphine assay is low (estimated at 0.003 and 0.002%, respectively), it remains a significant consideration given the likely high concentrations of these compounds in urine relative to the low cutoff of the buprenorphine assay. Neither amisulpride nor sulpiride was listed as potential sources of interference on the CEDIA data sheet when this work was performed. These findings highlight the importance of confirming immunoassay-positive buprenorphine results using a more selective analytical technique.

An automated method for the simultaneous measurement of azole antifungal drugs in human plasma or serum using turbulent flow liquid chromatography-tandem mass spectrometry.

Azole antifungal drugs are important in the prophylaxis and treatment of invasive aspergillosis. Therapeutic drug monitoring may be indicated to (1) monitor adherence, (2) guide dosage and (3) minimise the risk of drug-drug interactions and dose-related toxicity. TurboFlow(TM) technology offers online, automated sample preparation. An Aria Transcend(TM) TLX-II coupled with a TSQ Vantage(TM) MS was used. Centrifuged samples (25 µL) were mixed with internal standard solution (975 µL) and 30 µL injected directly onto a C18-P-XL TurboFlow column. Analytes were focussed onto a Phenomenex Gemini Phenyl analytical column and eluted using a methanol/water gradient (flow-rate, 0.8 mL/min). Analytes were monitored in selected reaction monitoring mode (two transitions per analyte, positive mode APCI). Calibration ranges were as follows: itraconazole, hydroxyitraconazole, and posaconazole 0.05-5.0 mg/L; voriconazole and fluconazole 0.1-10 mg/L. Total analysis time was 12 min. TurboFlow column recovery was >77% for all analytes. Calibration was linear (R (2) > 0.99) for all analytes. Inter- and intra-assay imprecision (% RSD) was <8% and accuracy (nominal internal quality control values) 90-105% for all analytes. The limit of detection was 0.01 mg/L for all analytes. No matrix effects were observed. This method is simple, robust and suitable for measuring these compounds at concentrations attained during therapy.

An automated method for the measurement of a range of tyrosine kinase inhibitors in human plasma or serum using turbulent flow liquid chromatography-tandem mass spectrometry.

Tyrosine kinase inhibitors (TKIs) are used to treat a number of cancers, including chronic myeloid leukaemia and hepatocellular carcinoma. Therapeutic drug monitoring (TDM) may be indicated to (1) monitor adherence, (2) guide dosage, and (3) minimise the risk of drug-drug interactions and dose-related toxicity. On-line, automated sample preparation provided by TurboFlow technology (ThermoFisher Scientific) in conjunction with the sensitivity and selectivity of tandem mass spectrometry (MS/MS) detection may be applied to the analysis of single drugs and metabolites. We report the use of TurboFlow LC-MS/MS for the analysis of nine TKIs and metabolites (imatinib, N-desmethylimatinib, dasatinib, nilotinib, erlotinib, gefitinib, lapatinib, sorafenib, sunitinib) in human plasma or serum for TDM purposes. An Aria Transcend TLX-II system coupled with a TSQ Vantage was used. Samples (50 µL) were vortex mixed with internal standard solution (150 µL imatinib-D(8), gefitinib-D(8), sunitinib-D(10), and nilotinib-(13)C (2) (15) N(2) in acetonitrile) and, after centrifugation 100 µL supernatant were injected directly onto a 50 × 0.5-mm Cyclone TurboFlow column. Analytes were focussed onto a 50 × 2.1-mm (3 µm) Hypersil GOLD analytical column and eluted with an acetonitrile/water gradient. Analytes were monitored in selected reaction monitoring mode (positive APCI). Total analysis time was 7 min without multiplexing. Calibration was linear (R(2) > 0.99) for all analytes. Inter- and intra-assay precision (in percent relative standard deviation, RSD) was <11 % and accuracy 89-117 % for all analytes. No matrix effects were observed. This method is suitable for high-throughput TDM in patients undergoing chronic therapy with TKIs and has been utilised in the analysis of clinical samples.

Basic drug analysis by strong cation-exchange liquid chromatography-tandem mass spectrometry: simultaneous analysis of amisulpride, and of metamfetamine and amfetamine in serum/plasma.

In the HPLC of basic drugs and metabolites, good efficiency and peak shape can often be attained using strong cation-exchange packings with isocratic 100% methanol eluents containing an ionic modifier at an appropriate pH* and ionic strength. Solvent extracts can be analysed directly, and use of ammonium acetate as modifier facilitates the use of atmospheric pressure chemical ionization (APCI)-tandem mass spectrometry, selected reaction monitoring mode. For the analysis of amisulpride and of metamfetamine/amfetamine in plasma (200 µL) after single oral doses in man, a column packed with Waters Spherisorb S5SCX (5 µm average particle size, 100 × 2.1 mm i.d.) was used with methanolic ammonium acetate (40 mmol/L, pH* 6.0, flow rate 0.5 mL/min) as eluent (35°C). Deuterated internal standards were used for each analyte. Detection was by positive-mode APCI. Responses for all analytes were linear over the calibration ranges. Intra-assay precision (RSD) was 2-18%, and inter-assay precision was 2-12%. The limit of detection was 0.5 µg/L for all analytes. No significant matrix effects or isobaric interferences were noted. The total analysis time was 7 min. Similar methodology can be applied to a wide range of basic analytes using MS/MS detection.

Inversion of penetrable obstacles from far-field data on narrow angular apertures

Reported in this paper are reconstructions of shape and material parameters of two-dimensional, homogeneous, acoustic, penetrable obstacles of arbitrary cross-sections which are immersed in an infinite, homogeneous ambience. Reconstructions are based on the far-field scattering patterns of multiple incident plane acoustic waves. For remotely acquired data, practical necessities require not only that the entire data collection region be less than 2 pi, but also that each received "sees" the object over as narrow an angular aperture as possible. The inversions presented here were obtained under such conditions. Two types of data were used for each incidence namely, "near-monostatic" fields of narrow angular apertures (the narrowest aperture reported is 2 degrees), and a "duostatic" geometry consisting of backscatter plus one other receiver angle. These data sets were acquired for a series of incident angles. The theoretical formalism for inversion is algebraic in nature, requires no integral equation, and possesses a number of advantages for the implementation of a Gauss-Newton type of inversion that was used in this study. Moreover, the algorithm is shown to be inheritently parallelizable.