Β-expectation Tolerance Intervals Research Articles

Microextraction based on liquid-solid phase transition of benzoic acid: Extraction of statins from human urine followed by chromatographic analysis

Herein, a microextraction method was reported based on the liquid-solid phase transition of benzoic acid to quantify two statins, namely lovastatin and simvastatin in authentic human urine. The principle of the method is based on the phase transition of benzoic acid by altering the pH of the sample solution enabling efficient dispersion and phase separation in one step. Due to the moderate melting point of benzoic acid, its solidification is performed at ambient temperature without the need for sample cooling. Various experimental parameters that affect the performance of the analytes (i.e. extractant type and its concentration, acid type and concentration, and sample volume) have been examined and optimized. The method was validated based on the total error concept. For this purpose, accuracy profiles were constructed in the concentration range of 100–5000 ng mL−1 while β-expectation tolerance intervals fell within ±15% demonstrating that 95% of future results will not exceed the defined bias limits. The intra-day and inter-day method precision was less than 4.7% and 4.3% for both analytes, while the limit of detection was 15 ng mL−1 for both analytes. It was also proved that the usage of benzoic acid is advantageous in minimizing the potential inter-conversion of the analytes during the acidification step of the extraction procedure. The green potential of the proposed analytical scheme was examined based on Green Analytical Procedure index. The proposed sample pretreatment technique proved to be a valuable tool offering selectivity and rapidness. The developed method was used for the analysis of real human urine obtained after the administration of statin-based pharmaceutical formulations.

Use of an Uncertainty Profile to Validate High-Performance Liquid Chromatography (HPLC) for the Simultaneous Determination of Statins in Synthetic Pharmaceutical Products

Here is reported a full validation of a high-performance liquid chromatography (HPLC) method for the determination of statins in a pharmaceutical product using the uncertainty profile. In addition, the uncertainty estimates based on the β-content, γ-confidence tolerance intervals are compared to those using the β-expectation tolerance interval via a graphical comparison. The results are useful in elucidating the applicability and flexibility of the uncertainty profile, which allows the analyst to choose the desired proportion of the results with a well-determined uncertainty. This approach allowed the determination of the validity of the statin assays. Four of the five statin dosages were shown to be accurate by demonstrating that more than 90% of the outcomes were compliant; however, rosuvastatin’s dosage was valid only by ensuring an 80% compliance rate. The uncertainty profile is a widely applicable strategy that may be used to evaluate analytical methods and compute the uncertainty in order to confirm suitability for use while providing reliable results.

Optimized LC-MS/MS quantification of tuberculosis drug candidate macozinone (PBTZ169), its dearomatized Meisenheimer Complex and other metabolites, in human plasma and urine

Tuberculosis, and especially multidrug-resistant tuberculosis (MDR-TB), is a major global health threat which emphasizes the need to develop new agents to improve and shorten treatment of this difficult-to-manage infectious disease. Among the new agents, macozinone (PBTZ169) is one of the most promising candidates, showing extraordinary potency in vitro and in murine models against drug-susceptible and drug-resistant Mycobacterium tuberculosis. A previous analytical method using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed by our group to support phase I clinical trials of PBTZ169. These plasma sample analyses revealed the presence of several additional metabolites among which the most prominent was H2PBTZ, a reduced species obtained by dearomatization of macozinone, one of the first examples of Meisenheimer Complex (MC) metabolites identified in mammals.Identification of these new metabolites required the optimization of our original method for enhancing the selectivity between isobaric metabolites as well as for ensuring optimal stability for H2PBTZ analyses. Sample preparation methods were also developed for plasma and urine, followed by extensive quantitative validation in accordance with international bioanalytical method recommendations, which include selectivity, linearity, qualitative and quantitative matrix effect, trueness, precision and the establishment of accuracy profiles using β-expectation tolerance intervals for known and newer analytes.The newly optimized methods have been applied in a subsequent Phase Ib clinical trial conducted in our University Hospital with healthy subjects. H2PBTZ was found to be the most abundant species circulating in plasma, underscoring the importance of measuring accurately and precisely this unprecedented metabolite. Low concentrations were found in urine for all monitored analytes, suggesting extensive metabolism before renal excretion.

A simple and green liquid chromatography method for the determination of ibuprofen in milk-containing simulated gastrointestinal media for monitoring the dissolution studies of three-dimensional-printed formulations.

A fast and green ultra-high-performance liquid chromatographic method was developed for the determination of ibuprofen in milk-containing simulated gastrointestinal media to monitor the dissolution of three-dimensional printed formulations. To remove interfering compounds, protein precipitation using methanol as a precipitation reagent was performed. The separation of the target analyte was performed on a C18 column using a mobile phase consisting of 0.05% v/v aqueous phosphoric acid solution: methanol, 25:75% v/v. Method validation was conducted using the total error concept. The β-expectation tolerance intervals did not exceed the acceptance criteria of±15%, meaning that 95% of future results will be included in the defined bias limits. The relative bias ranged between ─1.1 and +3.2% for all analytes, while the relative standard deviation values for repeatability and intermediate precision were less than 2.8% and 3.9%, respectively. The achieved limit of detection was 0.01μg/ml and the lower limit of quantitation was established as 2μg/ml. The proposed method was simple, and it required reduced organic solvent consumption following the requirements of Green Analytical Chemistry. The method was successfully employed for the determination of ibuprofen in real biorelevant media obtained from dissolution studies.

Green homogeneous liquid-liquid microextraction at high salinity conditions for the determination of nimesulide in human urine with the aid of experimental design

Green homogeneous liquid-liquid microextraction (HLLME) under high salinity conditions was proposed for the determination of nimesulide in human urine. The proposed analytical scheme consisted of the mixing of the appropriate volume of acetonitrile and sample followed by the addition of Na2SO4 solution to obtain phase separation. Following the extraction of the drug from the urine samples, high-performance chromatography-diode array detection (HPLC-PDA) was used for its determination. The main factors influencing the performance of the HLLME method were studied and optimized using a two-step experimental design (Plackett-Burman, Central Composite Design). The HLLME-HPLC-PDA method was systematically validated using the accuracy profiles. The detection limit was adequate and estimated to be 0.03 μg mL−1. The mean analytical bias (expressed as relative error) ranged between ─ 2.9–3.6% and the relative standard deviation (RSD) was better than 3.7%. Moreover, the β-expectation tolerance intervals were in the range of ±15% demonstrating that 95% of the results will fall within the defined bias limits. The ruggedness of the herein developed protocol was assessed using Monte-Carlo simulations followed by capability analysis. The proposed scheme was applied for the determination of the drug in real urine samples following the administration of pharmaceutical formulations that contained nimesulide.

Salt-Induced Homogeneous Liquid–Liquid Microextraction of Piroxicam and Meloxicam from Human Urine Prior to Their Determination by HPLC-DAD

A salt-induced homogeneous liquid–liquid microextraction (SI-HLLME) protocol combined with high-performance liquid chromatography–diode array detection is presented for the first time for the determination of piroxicam and meloxicam in human urine. The main parameters affecting the performance of the sample preparation protocol were optimized by means of a two-step experimental design (i.e., 2-level fractional factorial design and Box–Behnken design). Following its optimization, the proposed method was thoroughly validated in terms of the total error concept in order to take into consideration the random and systematic errors. For the target analytes, accuracy profiles were constructed, and they were used as graphical decision-making tools. In all cases, the β-expectation tolerance intervals complied with the acceptance criteria of ±15%, proving that 95% of future results will fall within the defined bias limits. The limits of detection were 0.02 μg mL−1 and 0.03 μg mL−1 for piroxicam and meloxicam, respectively. The relative standard deviations were lower than 4.4% in all cases, and the mean relative biases ranged between −5.7 and 3.4% for both drugs. The proposed scheme is simple and rapid, while it is characterized by high sample throughput. Moreover, SI-HLLME requires reduced sample and reagent consumption, according to the requirements of Green Analytical Chemistry.

Development and Validation of an HPLC-UV Method for the Dissolution Studies of 3D-Printed Paracetamol Formulations in Milk-Containing Simulated Gastrointestinal Media.

Herein, a simple and rapid HPLC method for the determination of paracetamol milk-containing biorelevant media is proposed. The separation of the analyte from the milk-containing biorelevant media was accomplished isocratically using a mobile phase containing 25 mM phosphate buffer (pH = 3.0) and methanol, 80:20, v/v at a flow rate of 1 mL min−1. Following a protein precipitation-based sample clean-up, a thorough investigation of the effect of the precipitation reagent (methanol, acetonitrile, 10% v/v trifluoroacetic acid solution) on the analyte recovery was performed. The matrix effect was assessed in each biorelevant medium by comparing the slopes of the calibration curves of aqueous and matrix-matched calibration curves. The method was comprehensively validated using the accuracy profiles. The β-expectation tolerance intervals did not exceed the acceptance criteria of ±15%, meaning that 95% of future results will be included in the defined bias limits. The relative bias ranged between −4.5 and +3.9% for all analytes, while the RSD values for repeatability and intermediate precision were less than 2.7% and 3.0%, respectively. The achieved limit of detection (LOD) was 0.02 μg mL−1 and the lower limits of quantitation (LLOQ) were established as 10 μg mL−1, which corresponded to 2% of the highest expected concentration of paracetamol. The proposed scheme was utilized for the determination of paracetamol in dissolution studies of its 3D-printed formulation in milk-containing biorelevant media.

HPLC Determination of Colistin in Human Urine Using Alkaline Mobile Phase Combined with Post-Column Derivatization: Validation Using Accuracy Profiles.

In this study, the development, validation, and application of a new liquid chromatography post-column derivatization method for the determination of Colistin in human urine samples is demonstrated. Separation of Colistin was performed using a core–shell C18 analytical column in an alkaline medium in order (i) to be compatible with the o-phthalaldehyde-based post-column derivatization reaction and (ii) to obtain better retention of the analyte. The Colistin derivative was detected spectrofluorometrically (λext/λem = 340/460 nm) after post-column derivatization with o-phthalaldehyde and N-acetyl cysteine. The post-column derivatization parameters were optimized using the Box–Behnken experimental design, and the method was validated using the total error concept. The β-expectation tolerance intervals did not exceed the acceptance criteria of ±15%, meaning that 95% of future results would be included in the defined bias limits. The limit of detection of the method was adequate corresponding to 100 nmol·L−1. The mean analytical bias (expressed as relative error) in the spiking levels was suitable, being in the range of −2.8 to +2.5% for both compounds with the percentage relative standard deviation lower than 3.4% in all cases. The proposed analytical method was satisfactorily applied to the analysis of the drug in human urine samples.

In situ synthesis of monolithic sol–gel polyethylene glycol-based sorbent encapsulated in porous polypropylene microextraction capsules and its application for selective extraction of antifungal and anthelmintic drugs from human urine

Herein, a monolithic sol–gel polyethylene glycol-based sorbent (PEG) has been in situ synthesized within two conjoined porous polypropylene microextraction capsules and subsequently applied for the microextraction of triazoles and benzimidazoles drugs from human urine. A cylindrical bar magnet was incorporated into the device, offering the ability of spinning when it is placed in a magnetic field resulting in a stand-alone “all-in-one” extraction device. Among other sorbent tested, the sol–gel PEG 300 coated microextraction capsules were found to be beneficial for the extraction of the target analytes. The analysis of the drugs was performed using high-performance liquid chromatography and photodiode array detection (HPLC-PDA). Detailed investigation of the main parameters that affect the extraction performance (i.e., dimensions of the microextraction capsules, sample volume, extraction time, ionic strength, stirring rate, elution time, type of eluent, volume of eluent) was conducted to find the optimum conditions for the extraction procedure. The method was thoroughly validated using the total-error concept. The β-expectation tolerance intervals did not exceed the acceptance criteria of ± 15% meaning that 95% of future results will be included in the defined bias limits. The lower limits of quantitation (LLOQs) of the method were adequate, corresponding to 0.1 and 0.25 μg mL−1 for triazoles and benzimidazoles, respectively. The mean analytical bias in the spiking levels was satisfactory, ranging between ─ 3.8 to + 6.7 % while the relative standard deviation (RSD) was lower than 5.6 %. Finally, the sol–gel PEG 300 encapsulated microextraction capsules were found to be reusable for at least 15 extraction cycles. The developed approach was successfully applied in the analysis of the selected drugs in urine samples.

Analytical quality-by-design optimization of UHPLC method for the analysis of octreotide release from a peptide-based hydrogel in-vitro

In this article, a UHPLC-PDA method has been developed using the quality-by-design (QbD) principles for the determination of the therapeutic peptide – octreotide – in its in vitro released samples. Due to the complexity of the peptide-based hydrogel matrix a reliable separation of the analyte from the matrix has to be performed. Risk assessment and multivariate analysis were employed for the method evaluation. Following method scouting towards the selection of appropriate and rapid UHPLC operative mode, quality risk assessment tools were applied to set the critical method parameters (CMPs) to be considered in screening phase. The effects of CMPs on critical method attributes (CMAs) were assessed further by means of a screening design. A response surface methodology was utilized to model CMAs as a function of the selected CMPs and the optimum separation conditions were additionally evaluated using desirability function. The method operable design region was complimented by establishment of a robust zone using Monte Carlo simulation and capability analysis. The method was validated in the range of 1 – 20 μg /mL using the accuracy profiles as a graphical decision-making tool. The β-expectation tolerance intervals was within the pre-set acceptance criteria of ± 10% meaning that 95% of future results will be included in the defined bias limits. The relative bias was varied between ─ 0.8% and 1.4% and the RSD values for repeatability and intermediate precision were below to 2.8% in all cases. The achieved limit of detection (LOD) and the lower limit of quantification (LLOQ) were adequate for the specific purpose and found to be 0.3 and 1 μg /mL, respectively. The developed method was successfully applied to the analysis of octreotide in in-vitro drug release samples obtained from peptide-based hydrogels.

Salting-out homogeneous liquid-liquid microextraction for the determination of azole drugs in human urine: Validation using total error concept.

A salting-out homogeneous liquid-liquid microextraction was proposed for the quantification of four azole drugs in human urine prior to high-performance liquid chromatography analysis. The procedure involved the mixing of the sample with acetonitrile in appropriate volumes followed by the addition of sodium sulfate solution in order to facilitate phase separation. The parameters influencing the extraction performance were studied and optimized using a two-step experimental design. The analytical procedure was thoroughly validated using the accuracy profiles as a graphical decision-making tool. The β-expectation tolerance intervals did not exceed the acceptance criteria of ±15% meaning that 95% of future results will be included in the defined bias limits. The limits of detection of the procedure were satisfactory, ranging between 0.01 and 0.03 μg/mL. The mean analytical bias in the spiking levels was satisfactory and ranged between -10.3 and 4.2% while the relative standard deviation was lower than 5.6%. Monte-Carlo simulations followed by capability analysis were employed to investigate the ruggedness of the sample preparation protocol. The developed method offers advantages compared to previously reported approaches for the same type of analysis including extraction efficiency and scaling down of the sample volume and extraction time.

Exploiting the capsule phase microextraction features in bioanalysis: Extraction of ibuprofen from urine samples

Undoubtedly, the exploration of new sample preparation techniques is in the forefront of research in bioanalysis. Capsule phase microextraction (CPME) is a recently introduced extraction technique based on the sol–gel sorbents which are incorporated into a porous tubular polymeric membrane. Herein, we exploited the advantageous features of CPME in biological matrices by utilizing sol–gel poly(tetrahydrofuran) (sol–gel PTHF) sorbent in CPME device for the extraction of ibuprofen (model compound) from urine samples. In CPME, both filtration and stirring mechanisms are merged into a unified extraction device allowing the direct extraction of the analytes from biological matrix without any pretreatment process. The main parameters (sample pH and volume, adsorption and elution time, volume of elution volume etc.) affecting the extraction efficiency of the CPME method were systematically investigated and optimized. The determination of ibuprofen was performed using UHPLC-PDA. The reported method was validated in terms of linearity, selectivity, accuracy, and precision, limit of detection and limit of quantification using the total error concept. Employing the accuracy profiles – as a graphical decision-making tool – the β-expectation tolerance intervals did not exceed the acceptance criteria of ± 15% meaning that 95% of future results will be included in the defined bias limits. The limit of detection (LOD) of the method was adequate, corresponding to 14 ng mL−1. The relative bias in the spiking levels was acceptable being in the range of −4.1 and +3.9% while the relative standard deviation (RSD) was lower than 4.6%. Moreover, the sol–gel sorbent integrated microextraction capsules were found to be reusable for at least 20 extraction cycles. The CPME protocol was successfully employed for the analysis of real urine samples obtained by a healthy volunteer. Finally, the developed CPME method was characterized by absence of matrix-effect and thus its application can be potentially expanded to other types of biological matrices.

Homogeneous liquid phase microextraction using hydrophilic media for the determination of fluoroquinolones in human urine using HPLC-FLD

Miniaturization of liquid-phase extraction is an increasingly emerging field of sample preparation aiming to reduce the solvent consumption and to protect the environment. In this paper, a homogeneous liquid phase microextraction using a hydrophilic extraction media in combination with a salt as phase-forming agent was developed for the determination of four fluoroquinolones (ciprofloxacin, norfloxacin, moxifloxacin and prulifloxacin) in human urine. Important parameters affecting the extraction performance including sample volume, organic solvent volume, salt concentration etc were systematically investigated and optimized. Multivariate experimental designs namely Plackett-Burman and Box-Behnken designs were sequentially applied for factor screening and optimization of the method parameters.The method was fully validated using the “total error” approach. Accuracy profiles – a graphical decision-making tool – were constructed using the results of the validation procedures. The β-expectation tolerance intervals did not exceed the acceptance criteria of ± 15%, meaning that 95% of future results will be included in the defined bias limits. The relative bias ranged between ─ 2.8 to 7.7 % for all analytes, while the RSD values for repeatability and intermediate precision were less 6.6%. The achieved limits of detection (LOD) were ranged between 3 and 11 ng mL−1 while the lower limits of quantitation (LLOQ) were established as 10 ng mL−1 for ciprofloxacin and norfloxacin and 100 ng mL−1 for moxifloxacin and prulifloxacin, respectively. The ruggedness of the microextraction procedure was assessed through Monte-Carlo simulations and capability analysis. The proposed approach improves the procedures proposed to date for the bioanalysis of fluoroquinolones in terms of efficiency, reduction of the sample volume and extraction time.

Exact equal-tailed β-expectation tolerance intervals for sample variances

The sample variance of the quality characteristic shows the variation of a product or process. When the precision is a concern, a tolerance interval (TI) for sample variances is needed. In this paper, we construct β-expectation TIs for the population of sample variances, assuming normality of the data, from the frequentist and Bayesian perspectives. The accuracy level of each TI, which is the probability that the actual coverage falls into a specified range, is calculated to evaluate the performance of the TI. Besides, the determination of the minimum number of subgroups and the minimum subgroup size is discussed to achieve the desired accuracy level. When the accuracy level, the number of subgroups and subgroup size are known, the maximum variation is also calculated to measure the goodness of the proposed TI. Comparison results show that a Bayesian TI outperforms the corresponding frequentist TI. A real example is used to demonstrate the applicability and implementations of the proposed TIs.

CBD oils on the Belgian market: A validated MRM GC-MS/MS method for routine quality control using QuEChERS sample clean up

Quality control of CBD oils on the Belgium market showed that the CBD content not always corresponds to the label claim. There is a pressing need to develop new analytical methods specifically developed to the assay of such oily samples. Analytical issues are, however, encountered for routine analyses due to the matrix complexity, high cost of cannabinoid standards and low Δ9-THC concentrations. An oily matrix could cause technical damages to analytical instruments and reduce the lifetime of the chromatographic columns. This paper proposes a procedure combining a sample cleanup by QuEChERS, removing the oily matrix, followed by a validated MRM GC-MS/MS method for the routine analysis of CBD oil samples. Eighteen CBD samples were selected on the Belgium market for analysis. This method allows the quantification of CBD, the legality check for the Δ9-THC content by a CBN standard and the screening of seven other cannabinoids namely CBN, CBDV, CBT, CBC, Δ8-THC, THCV and CBG. The method was validated at three concentration levels (0.5–1–2% (w/v)) for CBD and (0.05–0.1–0.2% (w/v)) for CBN. The detection limits for CBT, CBD, CBC, Δ8-THC, CBN and for the other cannabinoids of interest, were 10 and 14 ng/mL respectively. The accuracy profile values for CBD and CBN showed that the β-expectation tolerance intervals did not exceed the acceptance limits of ± 20%, meaning that 90% of future measurements will be included within this error range.

Uncertainty of Size-Exclusion Chromatography Method in Quality Control of Bevacizumab Batches

In addition to the analytical challenges related to the size and complexity of biopharmaceutical drugs, the inherent variability that arises due to their manufacturing process requires monitoring throughout the production process to ensure the safety and efficacy of the finished product. In this step, validation data should demonstrate that the process is controlled and reproducible, whereas the manufacturing process must ensure the quality and consistency of the product. For this, the manufacturer sets specification limits according with regulatory guidance. In such a situation, the comparison of different batches is required in order to describe and analyze the variability between them. However, it is unclear how great the variability of the analytical method would be or that in producing the batches. The estimation of the β-expectation tolerance intervals based on the variance components to account for both between-batch and within-batch variability was proposed as a specification limit to control the heterogeneity between batches at the time of manufacture and to verify whether batches meet specification limits. At this point, the variance components were computed by the maximum likelihood method using a linear random model. For this, the protein content, expressed as a percentage of the actual concentration relative to the claim value, and the dimer content (expressed as percentage) were used as critical quality attributes (CQAs) in the monitoring and control process. We used real data from six bevacizumab commercial batches.

Validation of plasma amino acid profile using UHPLC-mass spectrometer (QDa) as a screening method in a metabolic disorders reference centre: Performance and accreditation concerns

IntroductionAmino acid (AA) analysis in plasma is essential for diagnosis and monitoring of inborn errors of metabolism (IEM). The efficacy of patient management is governed by the rapidity of AA profile availability, along with the robustness of the method. French quality guidelines and progress made in analytical techniques have led biologists to develop AA profile exploration via mass spectrometry (MS). ObjectivesThe aim of this study was to validate an analytical method with a single quadrupole mass spectrometer (MS) and to suggest reference values in regard to French quality and IEM society recommendations. Design and methodsPlasma samples from patients were deproteinised and derivatised with AccqTag™ reagent. Analysis was performed by reverse-phase chromatography coupled to QDA detector. We evaluated accuracy, intra-days and inter-days precision and limit of quantification by the β-expectation tolerance interval method for 27 AA. Method comparison was performed with the standard method (ion exchange chromatography, IEC) on Jeol Aminotac® and to tandem MS. Reference values were established on AA concentrations of the cohort of patients who had no IEM. ResultsOur method allowed the separations of almost all amino acids with a total run time of 12 min. Separation of isoleucine and alloisoleucine was incomplete (R = 0.55) but without impact on biological interpretation. Precision, accuracy and quantification were satisfactory (intra-days coefficient of variation (CV) was <5%, inter-days precision CV <10% and accuracy <15%). The limits of quantification were validated between 1 and 900 µmol/L. Results were comparable between the new method and IEC. ConclusionUltimately, we validated a rapid method on plasma for quantifying 27 amino acids that can be used in routine practice, according to French quality laboratories and SFEIM (French Society of Inborn Error of Metabolism) recommendations. Furthermore, estimated reference values were similar to those found in published studies focusing on other methods. Despite a lower specificity compared to tandem MS, the simplicity and rapidity of our method are the main advantage of this technique and place it as a major tool in IEM diagnosis and management.

Development and Validation of an Automated Zone Fluidics-Based Sensor for In Vitro Dissolution Studies of Captopril Using Total Error Concept.

In the present research, a zone fluidics-based automated sensor for the analysis of captopril in in vitro dissolution samples is reported. Captopril is reacted under flow conditions with Ni(II) (10 mmol L−1) in alkaline medium (0.15% v/v NH3) to form a stable derivate, which is monitored spectrophotometrically at 340 nm. The chemical and instrumental parameters were carefully investigated and optimized. The validation of the developed method was performed in the range of 5 to 120% of the expected maximum concentration using the accuracy profiles as a graphical decision-making tool. The β-expectation tolerance intervals did not exceed the acceptance criteria of ±10%, which means that 95% of future results will be encompassed in the defined bias limits. The variation of the relative bias ranged between −2.3% and 3.5% and the RSD values for repeatability and intermediate precision were lower than 2.3% in all cases. The limit of detection (LOD), and the lower and the upper limit of quantification (LLOQ, ULOQ) were satisfactory and found to be 1%, 5% and 120% (corresponding to 0.6, 2.78 and 66.67 μg mL−1 in dissolution medium). The developed method was successfully applied for the analysis of captopril in dissolution tests of two commercially available batches.

Homogeneous liquid liquid extraction using salt as mass separating agent for the ultra high pressure liquid chromatographic determination of doxorubicin in human urine

In the present report, a homogeneous liquid liquid extraction using salt as mass separating agent was developed for the determination of the anticancer drug doxorubicin in human urine. Sample preparation is based on the usage of hydrophilic acetonitrile as extraction solvent and its phase separation under high salinity conditions. The analysis of doxorubicin and its epimer, epirubicin, (used as internal standard) was carried out by Ultra High Pressure Liquid Chromatography coupled to fluorimetric detection (λex/λem = 430/580 nm). Total sample analysis was accomplished in less than 15 min including the sample preparation and LC separation steps. The parameters affecting the extraction efficiency and method sensitivity were systematically investigated and optimized using experimental design. The method was validated in-house using the accuracy profiles as a graphical decision-making tool. The β-expectation tolerance intervals did not exceed the acceptance criteria of ± 15% meaning that 95% of future results will be included in the defined bias limits. The limit of detection (LOD) of the method was adequate corresponding to 10 ng mL−1. The mean analytical bias (expressed as relative recoveries) in the spiking levels was acceptable being in the range of 94.7 – 106.3% while the relative standard deviation (RSD) was lower than 5.1%. Experimental designs were built to examine the robustness of the UHPLC method. The proposed method has been satisfactorily applied to the analysis of the selected drug in human urine samples.

Controlling the Reproducibility of AC50 Estimation during Compound Profiling through Bayesian β-Expectation Tolerance Intervals

During drug discovery, compounds/biologics are screened against biological targets of interest to find drug candidates with the most desirable activity profile. The compounds are tested at multiple concentrations to understand the dose-response relationship, often summarized as AC50 values and used directly in ranking compounds. Differences between compound repeats are inevitable because of experimental noise and/or systematic error; however, it is often desired to detect the latter when it occurs. To address this, the β-expectation tolerance interval is proposed in this article. Besides the classical acceptance criteria on assay performance, based on control compounds (e.g., quality control samples), this metric permits us to compare new estimates against historical estimates of the same study compound. It provides a measure that detects whether observed differences are likely due to systematic error. The challenge here is that limited information is available to build such compound-specific acceptance limits. To this end, we propose the use of Bayesian β-expectation tolerance intervals to validate agreement between replicate potency estimates for individual study compounds. This approach allows the variability of the compound-testing process to be estimated from reference compounds within the assay and used as prior knowledge in the computation of compound-specific intervals as from the first repeat of the compound and then continuously updated as more information is acquired with subsequent repeats. A repeat is then flagged when it is not within limits. Unlike a fixed threshold such as 0.5log, which is often used in practice, this approach identifies unexpected deviations on each compound repeat given the observed variability of the assay.