Volumetric absorptive microsampling combined with impact-assisted extraction for hematocrit effect free assays.

AIM: Volumetric absorptive microsampling (VAMS) is a recent technology available for sampling and analyzing low blood volume. The present work describes the utilization of VAMS for the quantitation of naproxen and ritonavir in human blood using a novel bead-based impact-assisted extraction (IAE) procedure. RESULTS: Sampling volume accuracy of the VAMS device was independent of the blood hematocrit (HCT) level, however analyte recovery decreased with increasing HCT when extracted using ultrasonication. In contrast, IAE was unaffected by HCT, resulting in quantitative recovery for all levels evaluated. Precision and accuracy batches, as well as matrix effect evaluation, met acceptance criteria. CONCLUSION: The IAE procedure coupled with VAMS is immune to HCT biases affecting sampling volume and recovery.

Pharmacokinetic bioequivalence, safety and acceptability of Ornibel®, a new polymer composition contraceptive vaginal ring (etonogestrel/ethinylestradiol 11.00/3.474 mg) compared with Nuvaring® (etonogestrel/ethinylestradiol 11.7/2.7 mg).

OBJECTIVE: To show the clinical development of Ornibel® (ExeltisHealthcare, Spain) a contraceptive vaginal ring manufactured with a new polymer composition and containing etonogestrel/ethinylestradiol, compared to Nuvaring® (MSD, Spain). SUBJECTS AND METHODS: Randomised, single dose, 2-period, 2-sequence, 2-stage crossover, comparative bioavailability study conducted in 40 healthy female subjects. All subjects received both treatments for 28 days in each of two periods, separated by a 28 days washout. Ornibel® contains etonogestrel/ethinylestradiol 11.00/3.47 mg and Nuvaring® contains etonogestrel/ethinylestradiol 11.7/2.7 mg, both rings delivering 120/15 µg/day. For the calculation of pharmacokinetic parameters, 37 blood samples were collected up to 840 h after each ring insertion to quantify plasma concentrations of etonogestrel and ethinylestradiol using a validated MS/MS-HPLC. Safety was assessed by adverse events recording, clinical laboratory and vital signs and tolerability by vaginal examination. Acceptability was investigated by a 5-point scale questionnaire. RESULTS: Bioequivalence was demonstrated in the first stage as the 94.12% Confidence Intervals of the primary parameters laid within the 80-125% acceptance range for both etonogestrel (Cmax: 96.81-112.20%; AUC0-504h: 98.71-108.61%; AUC0-t: 100.14-109.10%) and ethinylestradiol. (Cmax: 105.91-120.62%; AUC0-504h: 105.47-114.59%; AUC0-t: 108.31-117.61%). During the first day of use a burst effect was observed with Nuvaring®, with significantly higher level of ethinylestradiol (Cmax0-24h ratio: 78.34%, 94.12CI: 73.55-83.45%). Both products were well tolerated and accepted, without significant differences between them. CONCLUSION: Ornibel® is bioequivalent to Nuvaring® in terms of efficacy, safety, tolerability and acceptability. The new polymer composition provides Ornibel® with more stability and gradual hormonal release during the first day of use, particularly for ethinylestradiol.

Discovery of a novel trimebutine metabolite and its impact on N-desmethyltrimebutine quantification by LC-MS/MS.

BACKGROUND: A failure in incurred sample reanalysis (ISR) for N-desmethyltrimebutine (NDMT), during the analysis of a trimebutine-containing drug GIC-1001 Phase I study, led to the discovery of a never-before reported metabolite of trimebutine. RESULTS: A positive bias for NDMT during the ISR and post-reconstitution stability evaluations indicated the presence of an unstable metabolite of NDMT. Precursor ion scans performed on freshly extracted samples enabled the identification of this metabolite to be the NDMT glucuronide conjugate and its fragmentation pattern suggested that the glucuronide moiety was attached at the N-terminal of NDMT. CONCLUSIONS: An acidification step was introduced in the extraction procedure to completely hydrolyze the glucuronide and measure the total NDMT in plasma, rendering this method a successful fit-for-purpose assay.

Elimination of isobaric interference and signal-to-noise ratio enhancement using on-line mobile phase filtration in liquid chromatography/tandem mass spectrometry.

RATIONALE: Liquid chromatography/tandem mass spectrometry (LC/MS/MS) instruments are selective and sensitive but can still be affected by isobaric interference or chemical noise arising from multiple sources such as the mobile phase. In this study, a high-performance liquid chromatography (HPLC) on-line mobile phase filtration setup is described and used to remove interference to allow better detection of the analyte of interest. METHODS: For instance, a filtration device containing a chemical sorbent is installed at the HPLC outlet of the aqueous solvent pump A or the organic solvent pump B. This manuscript reports different case scenarios under reversed-phase and HILIC separations either in positive (ESI(+)) or negative electrospray ionization (ESI(-)) mode using selected reaction monitoring (SRM) scans as well as additional Q1 MS scans. RESULTS: The filtration of the aqueous effluent of the mobile phase using a porous graphitic carbon filter eliminated the isobaric interferences and improved the detectability of gestodene and perindopril-D4. Also, a strong cation-exchange guard column installed at the acetonitrile outlet pump was found helpful on reducing the baseline intensity and improving significantly the signal-to-noise ratio (S/N) of methenamine. Moreover, the on-line mobile phase filtration was efficient at removing chemical background ions in full scan mode. CONCLUSIONS: This strategy demonstrated its usefulness by removing co-eluting isobaric interference, and reducing chemical background ions from the mobile phase, while drastically improving S/N.

Choosing the appropriate matrix to perform a scientifically meaningful lipemic plasma test in bioanalytical method validation.

Laurence Mayrand-Provencher has obtained a Master of Science in Chemistry from Université de Montréal. With over 3 years of experience as a scientist in the bioanalysis industry, he is now a scientist in method development at Algorithme Pharma. His experiences have led him to conduct robust and effective method development of bioanalytical assays, specifically in the LC-MS/MS field. Many regulatory agencies include in their guidelines the need to investigate the effect of lipemic plasma on the reliability of the data as part of a bioanalytical assay validation. Lipids can cause matrix effect, specificity and recovery issues, which can potentially lead to inaccurate data if left unaccounted for. However, finding the appropriate matrix type to be used to perform a lipemic plasma test is a major challenge, as the differences between those commercially available are not well known. The work reported herein describes the differences in lipid content between normal plasma, synthetic lipemic plasma mixes, and two types of natural lipemic plasma. The results obtained show that natural plasma with high triglycerides content should be used to perform a scientifically meaningful lipemic plasma test.

Impact of organic solvent additive on the integrity of plasma samples in bioanalysis by LC-MS/MS.

BACKGROUND: Matrix effects are one of the major drawbacks of ESI-MS/MS. It is majorly caused by lipids in plasma, which can be overcome by using different extraction techniques. RESULTS: In this investigation, a major matrix effect was observed in samples containing a co-administered drug. Unknown compounds appeared over time in the human plasma samples spiked with co-administered drug creating major ion suppression. The changes in matrix integrity were associated with the organic solvent content in the plasma samples. CONCLUSION: The amount and type of organic solvent added to human plasma along with the storage conditions must be carefully determined during method development in order not to impact quantitation.

Use of polarity switching for the simultaneous bioanalysis of analytes with three orders of magnitude difference in concentration by LC-MS/MS.

BACKGROUND: The challenge of quantifying two compounds in a single assay with drastic dynamic ranges is to obtain linearity without source or detector saturation at the mass spectrometer. RESULTS: In positive-ionization mode, the nonlinear relationships for Desmethyl Mebeverine Acid (DMAC) were demonstrated using three common strategies to overcome this issue: using offset voltage parameters, less-sensitive product ion or 13C mass SRM transitions. On the contrary, nonlinear relationships for DMAC were overcome if negative-ionization mode was used. Due to Mebeverine analytical LLOQ, dilution was not suitable for a single assay of Mebeverine and DMAC. However, polarity switching in negative mode for DMAC was successfully found to compensate for the nonlinearity at the mass spectrometer while preserving Mebeverine linear regression model in positive mode. CONCLUSION: The polarity switching strategy has demonstrated the advantage of improving linearity for analytes having different ionization polarities and three orders of magnitude difference in concentration.

Severe impact of hemolysis on stability of phenolic compounds.

Eugénie-Raphaëlle Bérubé has obtained a Bachelor of Science in Biochemistry from Université du Québec à Montréal. She previously worked at the St-Lawrence Center of Environment, Canada, conducting biomarker analysis to measure the impact of contaminants on aquatic species. She has been working in the bioanalysis industry for the past 8 years at Algorithme Pharma, a CRO located in Laval, Canada, becoming a scientist in bioanalytical method development for the quantitation of pharmaceuticals in biological fluids. The presence of hemolyzed plasma samples can negatively impact preclinical and clinical sample analysis. During the method development of morphine, post-extracted instability issues were encountered in human hemolyzed plasma when compared with nonhemolyzed plasma (called normal plasma for simplicity). Investigation revealed that the presence of methemoglobin using a high pH reconstitution solution led to degradation of morphine over time. The degradation probably results from radical oxidation of the ionized phenolic group promoted by the presence of methemoglobin. Pseudomorphine, the product of oxidative dimerization of morphine, was observed as one of the degradation products in hemolyzed plasma. This hypothesis was extended to raloxifene, another phenol-containing compound. On the other hand, no instability was detected for drug products bearing a masked phenol group or carboxylic acid functionality. The issue of morphine instability was resolved by using a reconstitution solution at a pH below the pKa of the phenol moiety.

An alternative solution to overcome carryover issues in bioanalysis.

LC-MS/MS instruments are considered as the analytical technique of choice for bioanalytical assays. The LC-MS/MS technique acquired this status due to its robustness, specificity, sensitivity and good precision and accuracy. Nonetheless, with the use of these instruments, users also have to deal with some complex analytical problems such as carryover. Even if carryover is well known and widely discussed, it still remains a common issue in bioanalytical work. Although new autosampler instruments manufactured are specifically designed to fix the problem, the issue is still present because methods developed by the industry also change and evolve with the instrument. Requests for wider dynamic ranges and the use of a very sensitive detector that can reach very low LOQ are among the reasons. This article reviews the causes of carryover and proposes an autosampler needle seat backflush as a novel solution to address the issue.

Potentially new isobaric metabolite of fluvastatin observed by LC-MS/MS during incurred sample analysis.

BACKGROUND: Fluvastatin (Lescol(®)) is a racemic mixture of two erythro stereoisomers and metabolizes into two threo stereoisomers. A method was developed to chromatographically separate fluvastatin from its threo isomers to support a bioequivalence study. RESULTS: During incurred samples analysis, an additional peak sharing the same mass transition as fluvastatin was observed and interfered with fluvastatin's quantification. Consequently, the chromatographic conditions were modified in order to resolve fluvastatin and the interference. Extensive evaluations were performed to ensure that the interference is not a degradation product generated during sample processing. CONCLUSION: An isobaric compound of fluvastatin was discovered, which required chromatographic separation from fluvastatin to obtain a reliable bioanalytical method. The investigation performed suggests that the interference is produced in vivo and is a possible metabolite of fluvastatin.

Impact of sample hemolysis on drug stability in regulated bioanalysis.

Being regulated by agencies' guidances, the importance of a robust validated bioanalytical method is crucial as it may impact the validity of the pharmacokinetic data generated. During blood collection and processing, the presence of hemolyzed plasma samples may occur and as a result its impact must be investigated to ensure method robustness. Indeed, hemolyzed samples may affect the analyte recovery efficiency, as well as the chromatography. Furthermore, the stability of an analyte in hemolyzed plasma can be an issue as analyte degradation may occur. In this article we report two case studies where the analyte instability was a result of sample hemolysis. A description of the appropriate actions undertaken for the resolution of the issue will be discussed.

Pre-cut dried blood spot (PCDBS): an alternative to dried blood spot (DBS) technique to overcome hematocrit impact.

Quantification of analytes by Dried Blood Spots (DBS) on different paper cards has been extensively reported in the past several years. However, some factors limit the robustness of the precision and accuracy of DBS such as: hematocrit level, blood viscosity, analyte nature, spotting technique and spotting conditions. As such, the paper material used for DBS must meet strict quality control criteria to produce reliable quantification of drugs: uniformity, no chemical leaching and no chromatographic effect. To overcome these variables, especially the hematocrit impact, a modification of the traditional DBS, named Pre-Cut Dried Blood Spot (PCDBS), is presented. In contrast to the classical DBS technique, the new PCDBS procedure demonstrates no variation in response, within ±3%, independently of the hematocrit level or of the type of card used. The impact of the hematocrit level on the analyte recovery is discussed for both DBS and PCDBS approaches. Moreover, for quantification of naproxen by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS), the PCDBS technique was demonstrated to be as precise (%CV ≤3.1%) and accurate (%nominal between 95.4 and 104.4%) as the classical DBS procedure.

A practical approach to reduce interference due to in-source collision-induced dissociation of acylglucuronides in LC-MS/MS.

BACKGROUND: In LC-MS/MS, glucuronide conjugated metabolites may convert back to the parent drug due to in-source collision-induced dissociation (CID). RESULTS: During the bioanalysis of naproxen, it was noticed that naproxen acylglucuronide exhibited intense in-source CID to the naproxen [M+H](+) ion under positive ESI. However, no in-source CID of the acylglucuronide to the naproxen [M+NH(4)](+) adduct was observed. Furthermore, absolutely no in-source CID was detected under negative ESI. This phenomenon was not only observed for naproxen acylglucuronide but for eight other acylglucuronides compounds. CONCLUSION: We have shown that monitoring the parent drug [M-H](-) or [M+NH(4)](+) whenever possible could be an easy approach used by bioanalytical scientists to minimize the impact of in-source CID of acylglucuronides to the parent drug.

Challenges of developing a bioanalytical method for a macrolide immunosuppressant compound by LC-MS/MS.

The quantification of tacrolimus in human whole blood was developed by LC-MS/MS for a range of 50.0 to 50,000.0 pg/ml. Different challenges were faced during method development due to ion-suppression, lack of sensitivity and low recovery. The optimization of the extraction procedure played a crucial role as tacrolimus had to be isolated from red blood cells, to which it is strongly bound. Another particular challenge arose from the freeze-thaw stability where the extracted samples from fresh blood always showed a lower recovery. Finally, matrix effect was observed in some matrices over time, which resulted in a failed long-term stability in whole blood. In order to resolve the matrix effect issue, the sample procedure had to be improved. The final assay showed good recovery, low matrix effect, linearity, blood stability and good precision and accuracy.

Impact of methylation of acyl glucuronide metabolites on incurred sample reanalysis evaluation: ramiprilat case study.

BACKGROUND: Reanalysis of incurred samples showed that the bioanalytical method for the quantification of ramipril and ramiprilat was generating irreproducible results for ramiprilat. RESULTS: An additional peak interfering with ramiprilat was observed in the incurred samples but not in the calibrant and quality control samples. A similar interference was detected for ramipril, but it was chromatographically separated. Interferences were produced during sample preparation, which involves strong cation exchanger cartridges. The interfering products corresponded to the methylation of ramipril and ramiprilat glucuronide. CONCLUSION: Following this discovery, a reproducible method was developed and successfully validated for ramipril and ramiprilat. Additional stability tests were performed in the presence of glucuronide and diketopiperazine metabolites of ramipril and ramiprilat to demonstrate the method specificity.

Impact of oxcarbazepine sulfate metabolite on incurred sample reanalysis and quantification of oxcarbazepine.

BACKGROUND: Recently, incurred sample reanalysis (ISR) has become a requirement in bioanalysis. The general guidance recommends investigating ISR failure to evaluate the suitability of an analytical method. In the case of acceptable ISR evaluation, there were no precise recommendations for further testing when sporadic values were obtained. RESULTS: The ISR evaluation performed during a bioequivalence study for the anticonvulsant drug oxcarbazepine showed acceptable ISR results, but one particular chromatographic anomaly led to a thorough investigation. The finding of these tests showed that an oxcarbazepine phase II metabolite occasionally co-eluted with the drug and impacted oxcarbazepine's quantitation through in-source conversion. CONCLUSION: This paper demonstrates the necessity of rigorous interpretation of ISR results and close monitoring of all subject sample results.

Reliable procedures to evaluate and repair crosstalk for bioanalytical MS/MS assays.

Louis-Philippe Morin is a senior instrument application specialist at Algorithme Pharma, a CRO located in Laval, Canada. He has been working in the bioanalysis industry for the past 10 years where he became a subject matter expert in analytical instrumentation, especially in the MS field. His responsibilities in his current position are to optimize the workflow of the laboratory and to find new procedures, or approaches, to fix complex analytical problems. Louis-Philippe's expertise acquired over the years has led him to multiple publications regarding instrumentation. LC-MS/MS is the analytical technique of choice for the quantification of drugs in biological fluids. In recent years, MS/MS detection has been impacted by the rapid evolution of bioanalysis industry requirements. The availability of fast chromatographic systems, the demand for wider dynamic ranges and the extensive use of stable isotope-labeled internal standards in bioanalysis has pushed some triple quadrupole detectors to their limits of operation. Consequently, this situation has led to a re-evaluation of the problem of crosstalk as a potential cause of issues in bioanalysis. In this article, the importance of crosstalk verification on the MS/MS instrument will be demonstrated. Additionally, procedures to identify, evaluate and fix possible crosstalk issues during bioanalytical assays on MS/MS instruments are proposed.

Elimination of LC-MS/MS matrix effect due to phospholipids using specific solid-phase extraction elution conditions.

BACKGROUND: The objective is to find elution conditions using different solid-phase extraction chemistries to reduce the amount of phospholipids in plasma extract, which is associated with matrix effect in LC-MS. RESULTS: Phospholipids' recovery was reduced by decreasing the eluents' methanol content applied to silica-based and polymer-based reversed-phase solid-phase extraction cartridges while 100% acetonitrile applied to the silica-based cartridges drastically minimized the phospholipids' elution. Silanol interactions are involved in the increased retention of phospholipids with silica-based reversed-phase cartridges when using high percentages of ACN in eluents. The bioanalytical usefulness of these findings was confirmed by successful extraction recovery of pharmaceutical compounds. CONCLUSION: Combinations of specific eluents and reversed-phase solid-phase extraction cartridges were found to prevent matrix effect due to phospholipids.

Determination of naproxen using DBS: evaluation & pharmacokinetic comparison of human plasma versus human blood DBS.

BACKGROUND: Dried blood spots (DBS) sampling is a well-known technology for qualitative determination such as DNA analysis and screening of newborn metabolic disorders. The scientific community has recently expressed interest in applying the DBS technique for quantitative determination of drugs in biological fluid. RESULTS: Two new bioanalytical assays were developed and validated for the determination of naproxen in human plasma and in DBS samples using liquid chromatography coupled with tandem MS. Furthermore, plasma and DBS clinical samples were collected from four subjects enrolled as part of a bioequivalence study. Concentration data for plasma and DBS samples were determined and pharmacokinetic (PK) profiles in plasma and in DBS samples were compared. CONCLUSIONS: A strong correlation between PK data obtained by the DBS and conventional plasma method was observed, which makes DBS a valuable technique for further naproxen bioavailability and PK investigations and studies.