Omnia A. Ismaiel

Investigation of endogenous blood plasma phospholipids, cholesterol and glycerides that contribute to matrix effects in bioanalysis by liquid chromatography/mass spectrometry.

Matrix effects caused by compounds endogenous to the biological sample are a primary challenge in quantitative LC/MS/MS bioanalysis. Many approaches have been developed to minimize matrix effects such as optimization of sample extraction procedures and use of isotopically labeled internal standards. Unexpected matrix components may still remain undetected, however, because of the selective mass transitions monitored during MS/MS analysis. Glycerophosphocholines are the major phospholipids in plasma that have been widely shown to cause significant matrix effects on electrospray ionization efficiencies for target analytes. The purpose of this work was to investigate potential matrix effects resulting from different endogenous lipid classes, including phospholipids, acylglycerols and cholesterols, in order to establish a library for the relative presence of these components in biological sample extracts obtained by commonly used sample preparation techniques. Thirteen compounds were selected which were representatives of eight phospholipids classes, mono, di, triacylglycerols, cholesterol and cholesterol esters. Post-column infusion experiments were carried out to compare relative ion suppression effects of these compounds. Chlorpheniramine and loratadine were selected as model test analytes. A Concentration Normalized Suppression Factor (%CNSF) was defined to allow comparison of ion suppression effects resulting from different endogenous lipids according to their typical concentrations in human plasma and erythrocytes. A simple LC/MS/MS method was developed to monitor these endogenous components in sample extracts and their extraction recoveries from a plasma pool were compared using protein precipitation, liquid-liquid extraction, supported-liquid extraction, solid phase extraction and Hybrid SPE-precipitation methods. Endogenous lipid components other than GPChos, such as cholesterols and triacylglycerols, may result in significant matrix effects and should be monitored during method development. No single extraction procedure was efficient in removing all of the various lipid components. Use of the results presented here, along with a consideration of analyte chemical structure, the type of matrix and the type of sample preparation procedure, may help a bioanalytical scientist to better anticipate and minimize matrix effects in developing LC/MS/MS-based methods.

Monitoring phospholipids for assessment of ion enhancement and ion suppression in ESI and APCI LC/MS/MS for chlorpheniramine in human plasma and the importance of multiple source matrix effect evaluations.

Biological matrix effects are a source of significant errors in both electrospray (ESI) and atmospheric pressure chemical ionization (APCI) LC/MS. Glycerophosphocholines (GPChos) and 2-lyso-glycerophosphocholines (2-lyso GPChos) are known to fragment to form ions at m/z 184 and m/z 104, respectively. Phospholipids were used as markers to evaluate matrix effects resulting in both ion suppression and enhancement using ESI and APCI modes in the determination of chlorpheniramine in human plasma. Results revealed that GPChos and 2-lyso GPChos demonstrated very low ionization efficiency in the APCI mode, post-column infusion experiments were performed to confirm that suppression and enhancement matrix ionization effects coincided with the elution profiles of the phospholipids. The mean matrix effect for chlorpheniramine using APCI was 75% less than the mean matrix effect in ESI, making APCI the ionization method of choice initially even though the absolute response was lower than in the ESI mode. The resulting APCI method showed acceptable results according to the FDA guidelines; however, a multiple source relative matrix effects study demonstrated variability. It was concluded that an absolute matrix effects study in one source of biological fluid may be not sufficient to ensure the validity of the method in various sources of matrix. In order to obviate the multiple matrix source variability, we employed an isotopically labeled internal standard for quantification of chlorpheniramine in the ESI mode. An additional validation was completed with the use of chlorpheniramine-d(6) as the internal standard. This method met all acceptance criteria according to the FDA guidelines, and the relative matrix affects study was successful.

Determination of octreotide and assessment of matrix effects in human plasma using ultra high performance liquid chromatography–tandem mass spectrometry

A selective UHPLC-MS/MS method for determination of the therapeutic peptide octreotide in human plasma was developed and validated. This assay used a UHPLC C(18) column with 1.7 μm particle size for efficient separation and an ion-exchange SPE for selective extraction. Octreotide and its labeled internal standard, [(13)C(6)Phe(3)] octreotide, were extracted from human plasma using a simple Oasis® WCX μElution SPE method and analyzed with a total chromatographic run time of 7.5 min. Matrix effects were studied during method development by direct monitoring of representative phospholipids. On-line removal of phospholipids using column switching and pre-column back-flushing was carried out to trap and remove any residual phospholipid matrix interferences. The UHPLC column provided baseline separation between the analyte and matrix peaks. The chromatographic conditions yielded optimal retention and excellent peak shape for both the analyte and internal standard. The assay was linear in the concentration range of 0.025-25.0 ng/ml, inter- and intra-assay precision and accuracy were within 6.1% and ±1.93%, respectively. Recovery was ∼73%. Post-extraction addition experiments showed that matrix effects were less than 4%. This method for octreotide in human plasma has been validated and utilized to support of clinical pharmacokinetic studies.

Assessment of matrix effects and determination of niacin in human plasma using liquid–liquid extraction and liquid chromatography–tandem mass spectrometry

A simple, sensitive and rapid liquid-liquid extraction method for the analysis of nicotinic acid (niacin) and its labeled internal standard nicotinic acid-d4 (niacin-d4) in human plasma was developed and validated. The analyte and its internal standard were isolated from acidified plasma using a single liquid-liquid extraction procedure with methyl-t-butyl ether. The extracted samples were analyzed by liquid chromatography-tandem mass spectrometry in positive electrospray ionization mode with multiple reaction monitoring. The calibration curves were linear in the measured range between 5 and 1000 ng/mL and the limit of detection was calculated as 122 pg/mL. The method required 250 microL of human plasma and the total run time between injections was 3.5 min. Matrix effects were assessed by post-column infusion experiments, phospholipids monitoring and post-extraction addition experiments. The extraction of phospholipids and niacin from plasma was studied under acidic, neutral and basic conditions. Acidic conditions were optimal for both the recovery of niacin and the removal of phospholipids; the degree of matrix effects for niacin was determined to be 2.5%. It was concluded that effective removal of matrix components can overcome low recovery issues associated with liquid-liquid extractions of polar analytes.

Green Analytical Chemistry, Opportunities for Pharmaceutical Quality Control

This review article summarizes the opportunities for utilizing the green analytical chemistry (GAC) techniques and principles in the field of quality control (QC) of pharmaceuticals. Green analytical chemistry is considered a branch of the green chemistry based on the principles overlapping with the goals of sustainable development. General definitions of quality and quality control, the principles of GAC, proposals for greener sample pretreatment and greener chromatographic method of analysis applied in QC laboratories are discussed herein. The main goal is to achieve more eco-friendly analysis in QC laboratories through different strategies and techniques, replace toxic reagents, and modify or replace analytical methods and/or techniques with safer ones, making it possible to dramatically reduce the amounts of reagents consumed and waste generated.

Investigation of endogenous blood lipids components that contribute to matrix effects in dried blood spot samples by liquid chromatography-tandem mass spectrometry

Dried blood spot (DBS) sampling coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a rapidly developing approach in the field of biopharmaceutical analysis. DBS sampling enables analysis of small sample volumes with high sensitivity and selectivity while providing a convenient easy to store and ship format. Lipid components that may be extracted during biological sample processing may result in matrix ionization effects and can significantly affect the precision and accuracy of the results. Glycerophosphocholines (GPChos), cholesterols and triacylglycerols (TAG) are the main lipid components that contribute to matrix effects in LC-MS/MS. Various organic solvents such as methanol, acetonitrile, methyl tertiary butyl ether, ethyl ether, dichloromethane and n-hexane were investigated for elution of these lipid components from DBS samples. Methanol extracts demonstrated the highest levels of GPChos whereas ethyl ether and n-hexane extracts contained less than 1.0 % of the GPChos levels in the methanol extracts. Ethyl ether extracts contained the highest levels of cholesterols and TAG in comparison to other investigated organic solvents. Acetonitrile is recommended as an elution solvent due to low lipid recoveries. Matrix effects resulted from different extracted lipid components should be studied and assessed carefully in DBS samples.

Development and optimization of on-line 2-dimensional chromatographic approaches for eliminating matrix effects and improving bioanalysis of peptides in human plasma using UHPLC-MS/MS

Online 2-dimensional chromatographic approaches for eliminating matrix effects and optimizing bioanalysis of peptides using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) were studied. Three therapeutic peptides (octreotide, desmopressin, and vasopressin) were selected as model analytes. Human plasma was precipitated with acetonitrile; peptides were analyzed on C(8), C(18), Phenyl and HILIC ACQUITY UPLC columns. For simpler online clean-up applications, a C(18) pre-column was coupled to the analytical column via a switching valve. For more complex heart-cutting applications, two analytical columns were used with optional online dilution to refocus the analyte peaks prior to the second dimension separation. This allows the use of MS incompatible mobile phases, such as TFA, in the first dimension separation. Online clean-up effectiveness was investigated by monitoring phospholipids. Flushing direction, mobile phase composition, flow rate and transfer window were evaluated. Phospholipids were readily retained on reversed-phase columns, and the peptides were reproducibly transferred, individually or as a group, to the second column using appropriate transfer windows. The best peak shapes were obtained when the second dimension column was more retentive (e.g. C(18) vs. C(8)). However, C(8) to HILIC gave broad unresolved peaks due to mobile phase mismatch. Trapped phospholipids were efficiently removed from either guard columns or first dimensional columns by forward- or back-flushing at high flows; however, back-flushing was more efficient with lower flow rates on larger columns.

Development of a Liquid Chromatography-Negative ESI-Tandem Mass Spectrometry Method for Ibuprofen with Minimization of Matrix Effects Associated with Phospholipids

Abstract Phospholipids are known to cause matrix ionization effects during LC/MS/MS analysis of biological samples. Matrix effects involving endogenous phospholipids in the negative ESI mode have been investigated in this work for the determination of ibuprofen in human plasma. Glycerophosphocholines (GPChos) and 2-lysoglycerophosphocholines (2-lyso GPChos) were monitored as markers for endogenous matrix components. Various extraction solvents were evaluated to assess their abilities to remove phospholipids from plasma samples to minimize matrix effects. Post-column infusion experiments were applied to chromatographically resolve matrix effects that resulted from endogenous phospholipids. The resulting method was validated and linearity was obtained over a concentration range of 50 to 10,000 ng/mL.

Simple Spectrophotometric and Conductometric methods for Determination of Gemifloxacin in Pure, Pharmaceutical Dosage Form and Human Urine

Article history: Received on: 17/09/2015 Revised on: 09/01/2016 Accepted on: 03/03/2016 Available online: 28/12/2016 Two Simple, accurate, precise, and rapid spectrophotometric and conductometric methods were developed for the estimation ofgemifloxacin (GEM) in pharmaceutical dosage forms and biological human urine. Method A: A spectrophotometric method is based on the oxidation reaction between phosphomolybdic acid (PMA) and GEM to form molybdenum blue (Mo +5 ). Beer , s law was obeyed in the concentration range of (5-27 μg/ml). The correlation coefficient ( 2) for the studied drug was found to be 0.9999. The molar absorptivity ( ), Sandells sensitivity, limit of detection (LOD), and limit of quantitation (LOQ)were also calculated. Method B: A conductometric method is based on formation of an ion associate with PMA. It involves direct titration with PMA in the range of 1-20 mg. The precipitate obtained by ion pairing GEM with PMA has been spectroscopically characterized using IR spectroscopy. The method was successively applied to pharmaceutical formulations containing GEM. The results obtained were favorably compared with those obtained using the reported method.

Determination of Estragole in Pharmaceutical Products, Herbal Teas and Herbal Extracts Using GC-FID -

Article history: Received on: 13/01/2016 Revised on: 17/03/2016 Accepted on: 22/06/2016 Available online: 28/12/2016 Recent concerns about the potential carcinogenicity of estragole necessitate accurate determination of estragole in different products and extracts. GC-MS method has been used for characterization of Fennel extract; Estragole was found to constitute more than 65% of the extract. Accurate and simple method for the determination of estragole in pharmaceutical products, herbal teas and herbal extracts was developed using GCFID technique and p-anisaldehyde as an internal standard. Target analyte was extracted from different matrices by applying various extraction procedures such as hydro-distillation and ultrasound-assisted extraction. The hydro-distillation technique provided higher amounts of extracted estragole compared to ultrasound-assisted extraction. The calibration curve showed excellent linearity over a concentration range of 0.1–10 mg/ml with a correlation coefficient of 0.9997. Accuracy of back calculated calibration standards were within ±7.3 %. Precision and accuracy of quality control samples were within ± 9.0 %. Estragole levels were accurately measured in Fennel fruits, Chinese and Japanese Star Anise, Sekem ® teabags for cough, Baby Calm® teabags, Balsam®, Guava® syrup for cough and Aqua ream® syrup for diarrhea. Accurate concentrations of estragole should replace the non-specific label information found on most of the tested products.