The development of bioanalytical methods has become more and more challenging over the past years due to very demanding requirements in terms of method reliability, sensitivity, speed of analysis and sample throughput. LC-MS/MS has established itself as a method of choice for routine analysis of biological materials. A development of such method consists of several steps including sample preparation and clean-up step, efficient chromatographic separation, sensitive and selective detection of analytes in complex matrices, a choice of convenient data processing and calibration approach and finally method validation. Each of these steps has its own constraints and challenges, which are discussed in detail in this review. Novel and modern approaches in sample preparation, chromatography and detection are especially emphasized. Attention is paid to proper calibration approach and matrix effects that can seriously affect method accuracy and precision.
- MeSH
- Time Factors MeSH
- Chromatography, Liquid economics instrumentation methods MeSH
- Mass Spectrometry economics instrumentation methods MeSH
- Humans MeSH
- Validation Studies as Topic MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
A new, rapid and effective ultra-high-performance liquid chromatography method with mass spectrometry detection is described for the separation and quantification of 8-hydroxy-2-deoxyguanosine, 8-hydroxyguanosine and creatinine in human urine. The present study uses an isotope-labelled internal standard ([15N]5-8-hydroxy-2-deoxyguanosine), a BIO core-shell stationary phase and an isocratic elution of methanol and water. Sample preparation of human urine was performed by solid-phase extraction (SPE) on Oasis HLB cartridges with methanol/water 50:50 (v/v) elution. Extraction recoveries ranged from 98.1% to 109.2%. Biological extracts showed high short-term stability. Several aspects of this procedure make it suitable for both clinical and research purposes: a short elution time of less than 3.2 min, an intra-day precision of 2.5-8.9%, an inter-day precision of 3.4-8.7% and low limits of quantification (27.7 nM for 8-hydroxyguanosine, 6.0 nM for 8-hydroxy-2-deoxyguanosine). Finally, simultaneous analysis of DNA and RNA oxidative stress biomarkers is a useful tool for monitoring disease progression in neurodegenerative disorders and cancer. Graphical abstract UHPLC-MS/MS analysis of DNA and RNA oxidative stress biomarkers.
- MeSH
- Biomarkers urine MeSH
- Deoxyguanosine analogs & derivatives urine MeSH
- DNA urine MeSH
- Adult MeSH
- Solid Phase Extraction methods MeSH
- Guanosine analogs & derivatives urine MeSH
- Creatine urine MeSH
- Humans MeSH
- Limit of Detection MeSH
- Young Adult MeSH
- Neoplasms urine MeSH
- Neurodegenerative Diseases urine MeSH
- Oxidative Stress MeSH
- RNA urine MeSH
- Tandem Mass Spectrometry methods MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Young Adult MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Validation Study MeSH
Biomonitoring of human exposure to reactive electrophilic chemicals such as ethylene oxide (EO) has been commonly based on the determination of adducts with N-terminal valine in blood protein globin, but a systematic search has also been undertaken to find surrogate markers enabling non-invasive sampling. Recently, N-(2-hydroxyethyl)-L-valyl-L-leucine (HEVL) has been identified as an ultimate cleavage product of EO-adducted globin in the urine of occupationally exposed workers. Herein, full validation of the analytical procedure consisting of solid-phase extraction of HEVL from urine samples (2 mL) followed by high-performance liquid chromatography-electrospray ionization-high-resolution mass spectrometry determination using deuterium-labeled HEVL as an internal standard (IS) is described. Method limit of quantitation is 0.25 ng/mL, and its selectivity is excellent as demonstrated by the invariable ratio of the qualifier and quantifier ion intensities across diverse urine samples and synthetic standard. The linear calibration model was applicable over the whole concentration range tested (0.25-10 ng/mL). The method accuracy assessed as a recovery of HEVL using a spiking experiment was 98-100%. Within-day precision of the method ranged from 1.8% to 3.0%, while the results from consecutive analytical runs conducted within 1 week or within 10-150 weeks differed in the range of 2.2-9.7%. The stability study on urine samples (-20°C up to 3 years, freeze-and-thaw up to 10 cycles) as well as on aqueous solutions (5°C up to 4 months) indicated no relevant changes in HEVL concentration (≤4%) over the time tested. Analytical responses of both HEVL and IS correlated with urinary creatinine as an index of matrix composition, but this matrix effect was mostly eliminated using the HEVL/IS peak area ratio, attaining the IS-normalized relative matrix effect <3%. In conclusion, the method complied successfully with the bioanalytical method validation criteria, making it a reliable tool for HEVL determination in human biomonitoring.
- MeSH
- Dipeptides * MeSH
- Ethylene Oxide * MeSH
- Globins MeSH
- Leucine MeSH
- Humans MeSH
- Reproducibility of Results MeSH
- Chromatography, High Pressure Liquid MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
A validated, highly sensitive, and selective HPLC method with MS-MS detection has been developed for quantitative determination of azithromycin (AZI) in human Na2EDTA plasma. Roxithromycin (ROX) was used as internal standard. Human plasma containing AZI and internal standard was ultrafiltered through Centrifree Micropartition devices and the concentration of AZI was determined by isocratic HPLC-MS-MS. Multiple reaction monitoring mode (MRM) was used for MS-MS detection. The calibration plot was linear in the concentration range 2.55-551.43 ng mL(-1). Inter-day and Intra-day precision and accuracy of the proposed method were characterized by R.S.D and percentage deviation, respectively; both were less than 8%. Limit of quantification was 2.55 ng mL(-1). The proposed method was used to determine the pharmacokinetic profile of AZI (250-mg tablets).
- MeSH
- Anti-Bacterial Agents chemistry blood MeSH
- Azithromycin blood MeSH
- Time Factors MeSH
- Edetic Acid blood MeSH
- Mass Spectrometry methods MeSH
- Humans MeSH
- Reference Values MeSH
- Reproducibility of Results MeSH
- Roxithromycin chemistry MeSH
- Sensitivity and Specificity MeSH
- Ultrafiltration MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Validation Study MeSH
Cell-based bioassays are very sensitive and allow integrative effect screening of the whole environmental sample, which is usually composed of a mixture of agonists and antagonists. Measured toxicity is usually expressed as a bioanalytical equivalent concentration. So far, it is not possible to distinguish which part of this value is caused by the agonists and which by the antagonists. In this article, we present a simple method to analyze the dose-response curve of a mixture and to determine an agonistic bioanalytical equivalent concentration: a concentration of a reference chemical that would elicit the same effect as do only agonists in an unknown mixture. The method has been validated using several artificially prepared mixtures of agonists and competitive antagonists measured in a recombinant yeast assay. No difference was observed between the calculated equivalent concentrations and the used concentrations of the agonist in the mixture.
- MeSH
- Biological Assay methods MeSH
- Endocrine Disruptors * MeSH
- Estradiol pharmacology MeSH
- Estriol pharmacology MeSH
- Fulvestrant pharmacology MeSH
- Environmental Pollutants toxicity MeSH
- Drug Interactions MeSH
- Receptors, Estrogen antagonists & inhibitors MeSH
- Saccharomyces cerevisiae drug effects MeSH
- Models, Theoretical MeSH
- Publication type
- Journal Article MeSH
The emerging use of qPCR and dPCR in regulated bioanalysis and absence of regulatory guidance on assay validations for these platforms has resulted in discussions on lack of harmonization on assay design and appropriate acceptance criteria for these assays. Both qPCR and dPCR are extensively used to answer bioanalytical questions for novel modalities such as cell and gene therapies. Following cross-industry conversations on the lack of information and guidelines for these assays, an American Association of Pharmaceutical Scientists working group was formed to address these gaps by bringing together 37 industry experts from 24 organizations to discuss best practices to gain a better understanding in the industry and facilitate filings to health authorities. Herein, this team provides considerations on assay design, development, and validation testing for PCR assays that are used in cell and gene therapies including (1) biodistribution; (2) transgene expression; (3) viral shedding; (4) and persistence or cellular kinetics of cell therapies.
- MeSH
- Genetic Therapy * MeSH
- Polymerase Chain Reaction MeSH
- Tissue Distribution MeSH
- Drug Development * MeSH
- Publication type
- Journal Article MeSH
5-Hydroxymethylfurfural (HMF) was analyzed in 17 botanical varieties of honey from 12 countries. A recently developed high-performance thin-layer chromatographic (HPTLC) method was limited because of increased matrix effects at higher honey sample loading. Therefore, the method was modified to achieve higher sensitivity and eliminate matrix interference by use of rectangular application combined with a focusing step. The HPTLC results were compared with results from the new spectrophotometric Reflectoquant hydroxymethylfurfural assay. Both methods had quantification limits of 4 mg kg(-1) and were suitable for rapid quantification of HMF in honey at the strictest regulated level of 15 mg kg(-1). Comparable results were obtained for the 17 honey samples, with a mean deviation of 2.9 mg kg(-1) (15%). The optimized HPTLC method was proved to be highly matrix-robust and was validated for the 17 different honey matrices (correlation coefficients ≥0.9994 (n = 6), mean intra-day precision 3.2% (n = 3 within a plate; n = 2 repeated within a day), mean inter-day precision 3.7% (n = 3), mean reproducibility over the whole procedure including sample preparation 4.1% (n = 2), and mean recovery 106.9% (n = 5 different concentrations; n = 4 different honey matrices). Recovery for a range of different application volumes, and thus for different honey matrix loading, differed by only ≤4.2%. HMF results when calculated by use of external calibration and by use of the standard addition method varied by 8.8%. Both revealed that any matrix effect was minor and that the original matrix interference problem was successfully solved.
- MeSH
- Furaldehyde analogs & derivatives analysis MeSH
- Chromatography, Thin Layer methods MeSH
- Food Contamination analysis MeSH
- Honey analysis MeSH
- Reproducibility of Results MeSH
- Spectrophotometry methods MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
A study of complex protein mixtures obtained from biological samples by MS demands proper purification and separation technique. The method of divergent flow isoelectric focusing (DF IEF) promises improvement of sample preparation in proteomic studies. DF IEF was carried out in a separation channel with increasing width. The channel was cut out from a polyester nonwoven web. DC voltage (800 V) was brought to two pairs of electrodes situated on the channel sides. Amphoteric compounds, including proteins, drift through the channel carried by flow (18-25 ml/h) in streamlines given by their isoelectric points. The pH gradient (3-10) and its stability during analysis have been monitored with colored low-molecular mass pI markers. Separated fractions were collected in ten microvials and further analyzed by MS. The suggested method was used for separation and purification of crude protein extract from barley grain, malt, and beer. Collected fractions of separated proteins were characterized by MALDI-MS. Desalting during IEF enhanced significantly the quality of mass spectra. It also simplified monitoring of post-translational modifications and protein changes occurring during malting and brewing. Results have shown the real potential of the suggested DF IEF device lay-out as an efficient preparative tool for separation and purification of complex protein mixtures for further analyses.
- MeSH
- Time Factors MeSH
- Equipment Design MeSH
- Electrodes MeSH
- Financing, Organized MeSH
- Isoelectric Focusing methods instrumentation MeSH
- Hydrogen-Ion Concentration MeSH
- Analytic Sample Preparation Methods methods MeSH
- Molecular Weight MeSH
- Proteins analysis MeSH
- Proteomics MeSH
- Reproducibility of Results MeSH
- Sensitivity and Specificity MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods instrumentation MeSH
Boldine belongs to the group of aporphine alkaloids isolated from Boldo tree. In contrast with numerous reports on the pharmacological effects of boldine, the data about its pharmacokinetics and biotransformation are scarce. No validated bioanalytical method of sufficient sensitivity has so far been described in the literature which could be used for quantification of boldine in various body fluids collected in pharmacokinetic studies. This work presents, for the first time, the assay for boldine in the plasma, bile and urine of rats. It includes liquid-liquid extraction/back-extraction of boldine, its chromatographic separation and sensitive fluorescence detection. Separation was carried out on a pentafluorophenyl core-shell column (Kinetex PFP, 150×3mm, 2.6μm) in gradient elution mode with solvent system consisting of an acetonitrile-ammonium formate buffer (5mM, pH=3.8). Fluorimetric detection (λEX=320nm, λEM=370nm) was used for quantitative work. Validation according to the EMEA guideline proved the assay LLOQ (0.1μmolL(-1)), linearity over a broad range of 0.1-50μmolL(-1), precision (intra- and inter-day CVs less than 4.5% and 6.1%, respectively) and accuracy (relative errors between -5.8% and 4.8%). In a pilot pharmacokinetic experiment, the concentration-time profiles were described for boldine (single i.v. bolus 50mgkg(-1)) in plasma and bile and cumulative excretion in urine was investigated. The major metabolites identified by means of LC-MS(n) were boldine-O-glucuronide, boldine-O-sulphate and disulphate, boldine-O-glucuronide-O-sulphate and N-demethyl-boldine-O-sulphate.
- MeSH
- Aporphines analysis chemistry pharmacokinetics MeSH
- Spectrometry, Fluorescence MeSH
- Rats MeSH
- Least-Squares Analysis MeSH
- Reproducibility of Results MeSH
- Sensitivity and Specificity MeSH
- Drug Stability MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Bile chemistry MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
