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【Objective】 To investigate the matrix effect on the determination of potency in Recombinant Human Coagulation Factor Ⅷ for Injection (rFⅧ). 【Methods】 Two different detection matrices were used to establish two methods for detecting the potency in Recombinant Human Coagulation Factor Ⅷ for Injection. And the matrix effect on the determination of potency was determined, including specificity, linearity, repeatability, accuracy and intermediate precision. 【Results】 As to the specificity, the recoveries of the two substrates at high vs low concentration level were 112% and 110% vs 104% and 109%, respectively. As to the linearity, in the range of (0.125-1.000) IU/mL, the correlation coefficient between concentration and coagulation time of standard/ sample was higher than 0.99. As to the accuracy/repeatability, the recoveries of two matrices was 104% and 102%, and RSD was 2.4% and 1.9%. As to the intermediate precision, personnel factor of two matrices was 0.72 and 0.23, date factor was 0.79 and 0.85, and RSD(for 12 times) was 4.2% and 3.0%. Comparison of two matrices was as follows: Deviation in test results of 6 batches of rFⅧ was all lower than 5%. There was no significant difference between two matrices. 【Conclusion】 The two matrices for potency detection show good performance including specificity, linearity, repeatability, accuracy, and intermediate precision. They are suitable for the determination of potency in rFⅧ products.
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Sample preparation is considered as the bottleneck step in bioanalysis because each biological matrix has its own unique challenges and complexity.Competent sample preparation to extract the desired analytes and remove redundant components is a crucial step in each bioanalytical approach.The matrix effect is a key hurdle in bioanalytical sample preparation,which has gained extensive consideration.Novel sample preparation techniques have advantages over classical techniques in terms of accuracy,automation,ease of sample preparation,storage,and shipment and have become increasingly popular over the past decade.Our objective is to provide a broad outline of current developments in various bioanalytical sample preparation techniques in chromatographic and spectroscopic examinations.In addition,how these techniques have gained considerable attention over the past decade in bioanalytical research is mentioned with preferred examples.Modern trends in bioanalytical sample preparation techniques,including sorbent-based microextraction techniques,are primarily emphasized.
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AlM: The Chinese medicinal herb Hanfangji is dried roots of Stephania tetrandra S. Moore (Family, Menispermaceae). Tetrandrine and fangchinoline are two major constituents of Hanfangji and these bisbenzyltetrahydroisoquinoline alkaloids possess anti - cancer and other pharmacological activities. To facilitate further pharmacodynamic investigation of these compounds, a pharmacokinetic investigation was performed in rats and in vitro. METHODS: Pharmacokinetics of tetrandrine and fangchinoline were characterized in rats p.o. or i.v. dosing an aqueous extract of Hanfangji or the individual compound. Unbound levels of systemic exposure to these two alkaloids were assessed using in vitro studies of plasma protein binding, blood-plasma partition, and lysosomal trapping. All the study samples were analyzed by liquid chromatography/mass spectrometry.RESULTS: We found two pharmacokinetic features of tetrandrine and fangchinoline. First, the two compounds had blood levels of systemic exposure substantially higher than the respective plasma levels of systemic exposure. Second, the two compounds exhibited significantly higher systemic exposure levels after p.o. dosing an aqueous extract of Hanfangji than the respective exposure levels after p.o. dosing the individual compound, at the same compound dose levels and under the same conditions for analytical measurement and the same conditions for animal study. Unbound fractions of tetrandrine and fangchinoline in rat plasma were 2%-5% and the concentrations of the alkaloids in rat erythrocytes were 5-times higher than those in rat plasma. Lysosomal inhibitor could block their trapping in lysosomes and significantly reduce their concentrations in HEK-293 cells. CONCLUSlON: The following pharmacokinetic aspects should be noted in pharmacodynamic investigation of tetrandrine and fangchinoline: extensive binding with plasma proteins, extensive binding with erythrocytes, and trapping by lysosomes of tissue cells substantially reduce the levels of unbound tetrandrine and fangchinoline in the systemic circulation.
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An Liquid chromatography tandem mass spectrometry (LC-MS/MS) technique is one of the best analytical methods for thequantification of drugs in biological samples. A stability-indicating analytical technique was developed for the quantitationof tapentadolin biological matrices as tapentadol with short runtime. Developed technique also suitable for bioavailabilitystudies in healthy rabbits. Separation of tapentadol and tapentadol-d3 were achieved from plasma sample with solid-phaseextraction and elution was processed with Luna-C18 (5 μ, 100 mm × 4.6 mm) stationary column with movable phase ratiocomprising 2-mM ammonium acetate buffer (pH-3.6) and acetonitrile in the proportion of 10:90 % V/V. Quantitationwas processed by processing the transitions of tapentadol and tapentadol-d3 at m/z 222.2 → 177.1 and 228.2 → 183.1,respectively, in positive ionization mode. Linearity was performed over the concentration range of 0.121 to 35.637 mg/ml(R2 > 0.99) without matrix effect (2.74%). The inter- and intra-day precision findings were within 8.62% and 11.38%,respectively. Stability data showed that the tapentadol was stable when it exposed to different stability conditions. Thistechnique was effectively applied to bioavailability studies of tapentadol in healthy rabbits
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BACKGROUND: Liquid chromatographyetandem mass spectrometry (LC-MSMS) for simultaneous analysis of multiple microbial secondary metabolites (MSMs) is potentially subject to interference by matrix components. METHODS: We examined potential matrix effects (MEs) in analyses of 31 MSMs using ultraperformance LC-MSMS. Twenty-one dust aliquots from three buildings (seven aliquots/building) were spiked with seven concentrations of each of the MSMs (6.2 pg/µl–900 pg/µl) and then extracted. Another set of 21 aliquots were first extracted and then, the extract was spiked with the same concentrations. We added deepoxy-deoxynivalenol (DOM) to all aliquots as a universal internal standard. Ten microliters of the extract was injected into the ultraperformance LC-MSMS. ME was calculated by subtracting the percentage of the response of analyte in spiked extract to that in neat standard from 100. Spiked extract results were used to create a matrix-matched calibration (MMC) curve for estimating MSM concentration in dust spiked before extraction. RESULTS: Analysis of variance was used to examine effects of compound (MSM), building and concentration on response. MEs (range: 63.4%–99.97%) significantly differed by MSM (p < 0.01) and building (p < 0.05). Mean percent recoveries adjusted with DOM and the MMC method were 246.3% (SD = 226.0) and 86.3% (SD = 70.7), respectively. CONCLUSION: We found that dust MEs resulted in substantial underestimation in quantifying MSMs and that DOM was not an optimal universal internal standard for the adjustment but that the MMC method resulted in more accurate and precise recovery compared with DOM. More research on adjustment methods for dust MEs in the simultaneous analyses of multiple MSMs using LC-MSMS is warranted.
Subject(s)
DOM 2,5-Dimethoxy-4-Methylamphetamine , Calibration , Dust , Mass Spectrometry , MethodsABSTRACT
<p><b>Background</b>Measuring total serum calcium is important for the diagnosis of diseases. Currently, results from commercial kits for calcium measurement are variable. Generally, the performance of serum calcium measurements is monitored by external quality assessment (EQA) or proficiency testing schemes. However, the commutability of the EQA samples and calibrators is often unknown, which limits the effectiveness of EQA schemes. The aim of this study was to evaluate the bias of serum calcium measurements and the commutability of processed materials.</p><p><b>Methods</b>Inductively coupled plasma mass spectrometry was applied as a comparative method, and 14 routine methods were chosen as test methods. Forty-eight serum samples from individual patients and 25 processed materials were quantified. A scatter plot was generated from patient samples, and 95% prediction intervals were calculated to evaluate the commutability of the processed materials and measurement bias at three concentration levels was used to determine the accuracy of routine assays.</p><p><b>Results</b>All assays showed high precision (total coefficient of variation [CV] <2.26%) and correlation coefficients (r > 0.99). For all assays, the mean bias for the 48 patient samples ranged from -0.13 mmol/L to 0.00 mmol/L (-5.61-0.01%), and the ranges for the three concentrations were -0.10-0.04 mmol/L (-5.71-2.35%), -0.14--0.01 mmol/L (-5.80--0.30%), and -0.19-0.04 mmol/L (-6.24-1.22%). The EQA samples, calibrators, and animal sera exhibited matrix effects in some assays; human serum pools were commutable in all assays; certificate reference materials were commutable in most assays, and only GBW09152 exhibited a matrix effect in one assay; and aqueous reference materials exhibited matrix effects in most assays.</p><p><b>Conclusions</b>Biases for most assays were within the acceptable range, although the accuracy of some assays needs improvement. Human serum pools prepared from patient samples were commutable, and the other tested materials exhibited a matrix effect.</p>
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Objective To establish a method for the determination of 8 heavy metal elements of Pb,Cd,As,Hg,Co,V,Se,Mo in iron dextran by inductively coupled plasma optical emission spectrometry (ICP-OES).Method Through selection of detection wavelengths,optimization of instrument parameters,verification of matrix effect interference and investigation of internal standard for correction of matrix effect interference,the 8 heavy metal elements were analyzed by ICP-OES.Results The recovery rate of the detected elements were increased from 83％-90％ to 94％-100％,after correction of matrix effect interference by internal standard method.The accuracy of the method were good.The spiked recoveries of the detected elements were from 94.3％ to 101.5％.The precision of the method were good (RSD ＜ 3.5％,n =6).The linearities of the detected elements were good,and the correlation coefficients were all greater than 0.999.The detection limits were from 0.22 to 8.54 ng/mL.The quantization limits were from 0.68 to 25.73 ng/mL.Conclusion The established method was accurate,sensitivity,rapid and reliable,which can be applied to the determination of contents of 8 heavy metal elements in iron dextran.
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Objective To establish a method for the determination of 8 heavy metal elements of Pb,Cd,As,Hg,Co,V,Se,Mo in iron dextran by inductively coupled plasma optical emission spectrometry (ICP-OES).Method Through selection of detection wavelengths,optimization of instrument parameters,verification of matrix effect interference and investigation of internal standard for correction of matrix effect interference,the 8 heavy metal elements were analyzed by ICP-OES.Results The recovery rate of the detected elements were increased from 83％-90％ to 94％-100％,after correction of matrix effect interference by internal standard method.The accuracy of the method were good.The spiked recoveries of the detected elements were from 94.3％ to 101.5％.The precision of the method were good (RSD ＜ 3.5％,n =6).The linearities of the detected elements were good,and the correlation coefficients were all greater than 0.999.The detection limits were from 0.22 to 8.54 ng/mL.The quantization limits were from 0.68 to 25.73 ng/mL.Conclusion The established method was accurate,sensitivity,rapid and reliable,which can be applied to the determination of contents of 8 heavy metal elements in iron dextran.
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Objective To provide a proper concentration of Internal Standard Solution by comparing five different degrees to the simultaneous analysis of 10 metals including V,Cr,Mn,Fe,Ni,Cu,As,Cd,Pb and Hg in Cardeniajasminoides.Method An Inductively Coupled Plasma-Mass Spectrometry method with Microwave-Assisted Digestion was developed with10,50,200,500,1 000 μg/L of Ge,In,Bi as internal standard.Result The degree of 500 μg/L concentration of internal stand solution can both correct the matrix effect and alleviate the contamination as the 1 000 μg/L.Conclusion With excellent detection limits,high sensitivity and wide linear range,the 500 μg/L internal standard may serve as reference in the determination of elements in other traditional Chinese medicine (TCM).
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Quantitative calibration strategy as an essential issue in laser ablation ICP-MS plays an important role for the guarantee of analytical accuracy.In this study, uncertainties of reference values in current available glass certified reference materials (NIST, MPI-DING and USGS) as well as the matrix effect among them were systematically evaluated.The results illustrated that NIST610 was better than other reference materials from aspect of reference value uncertainty.The matrix effect among NIST, MPI-DING and USGS reference materials was insignificant under our experimental conditions.A quantification strategy based on two reference materials (NIST610 and StHs6/80-G) and normalization to 100% (w/w) was proposed to avoid the insufficiency of single reference material strategy, which suffered the very low content and large uncertainty for some elements.The comparision of ML3B-G results obtained from three quantification strategies (single reference material NIST610, single reference material StHs6/80-G and two refterence materials) illustrated that the proposed strategy improved the analytical accuracy.Three reference materials (BCR-2G, CGSG-2 and KL-2G) were quantified with the proposed strategy, and almost all data matched well with reference value, meanwhile the data reported here could supplement the reference value database.
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A rapid method was developed for the determination of 9 organophosphorous flame retardants ( OPFRs) in textiles by ultra high performance liquid chromatography tandem electrospray ionization mass spectrometry ( UHPLC-MS/MS ) . Analytes in textile samples were extracted in a closed bottle at 50℃ with methanol by ultrasonic extraction for 30 min. And then, the extracted solution was injected after filtration and determined by UHPLC-MS/MS. Target analytes were separated by C18 column and methanol/water containing 5. 0 mmol/L NH4 COOH. The positive electrospray ionization and multiple reaction monitoring mode ( MRM) was utilized. The matrix effect (ME) could be compensated by adjusting the concentration of NH4COOH and the composition of the mobile phase after careful investigation with blank samples. An external standard calibration method was chosen to the quantitative analysis. Under the optimized conditions, method precision represent with relative standard deviations (RSDs) was 5. 2% (TOCP) -12. 5% (TRIS) for cotton, 6. 4%(TBEP)-13. 8% (TRIS) for polyester, and 6. 9% (TDCP) -14. 5% (TEPA) for wool samples. The recoveries for standard addition were in the range of 52 . 5% ( TEPA ) -116 . 4% ( TPP ) . The limits of quantification (LOQ, S/N=10) were between 1. 0 (TBP) and 10. 0 (TRIS) μg/kg.
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Objective] To discuss the applicability of internal standard in determination of cycla-mate in wine by HPLC-tandem mass spectrometry . [ Methods] After dilution wine sample was deter-mined by HPLC-tandem mass spectrometry and quantified by external and internal standard methods respec-tively. [Results] The results by the 2 quantitation methods differed 11.2%in average, and the maxi-mum relative deviation was 16.2%.And the result by external standard quantitation method proved to be consistent with that by the international standard GB/T 5009.97-2003. [ Conclusion] The difference in results is thought to originate from matrix effect in HPLC-tandem mass spectrometry and the improper in-ternal standard compound applied .
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Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is a widely used technique in the quantitative analysis of small molecules, part of peptides and proteins in biological matrix. When compared with LC, the LC-MS/MS posesses many advantages, such as high sample throughput, high sensitivity and resolutions, simple method development and simultaneous quantification of multiple components. At the stage of method development, the main practices for analysts are to validate analytical methods according to the regulated requirements. However, due to the quantitative mechanisms of LC-MS/MS, even though the used method is fully validated, analytical inaccuracy problem, which is the so called bioanalytical risk (pitfalls), may still exist, if we do not pay attentions to some details. In the present paper, the factors which may cause inaccuracy in quantitative analysis are summarized, especially those could not be found during routine method validation. Those factors include back transformation of unstable drug metabolites to analyte in the handling of biosamples, matrix effects, in-source conversion, and ion interference caused by ions with similar or same mass-to-charge ratio, etc. In addition to QA/QC oversight and strict method validation, the preventing strategies may contain the follows: selecting biosample processing method with high proficiency in eliminating matrix interferences, enhancing chromatographic separation, selection of suitable precursor-product ions and validation with incurred samples, etc.
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Os antidepressivos pertencem a uma importante classe de medicamentos investigados na toxicologia forense. Em casos de amostras provenientes de cadáveres, o intervalo entre o óbito e a obtenção da espécie biológica pode proporcionar a redistribuição postmortem destes fármacos. Com o objetivo de elucidar esse fenômeno, métodos analíticos foram desenvolvidos e aplicados utilizando sangue total (ST), humor vítreo (HV) e fígado. Para as amostras de ST e HV, o método de extração escolhido e validado foi a microextração em fase líquida (LPME) trifásica. Fibras ocas constituídas de polipropileno, com a extensão de 8 cm cada, foram tratadas com o solvente orgânico dodecano (fase orgânica), resultando em um membrana com permeabilidade seletiva. No lúmen destas fibras, adicionou-se ácido fórmico 0,1 mol/L (fase aceptora). Em frasco de fundo chato com 5 mL de capacidade, pipetou-se 3,5 mL de NaOH 0,1 mol/L (fase doadora) e 0,5 mL de ST ou HV. Ao término da extração, as amostras foram introduzidas no GC-MS, sem a necessidade de reações de derivatização. O estudo com ST contemplou os antidepressivos amitriptilina (AMI), nortriptilina (NTR), imipramina (IMI), desipramine (DES), clomipramina (CLO), desmetilclomipramina (DMC), fluoxetina (FLU) e norfluoxetina (NFL). Os limites de quantificação para estas substâncias ficaram inferiores aos níveis terapêuticos (20 ng/mL). As médias dos coeficientes de variação intradia e interdia foram, respectivamente, de 9,7 e 9,8%. As curvas de calibração apresentaram linearidade entre as concentrações de 20 até 1200 ng/mL. A validação do parâmetro integridade da diluição assegurou a mensuração de quantidades superiores ao limite apresentado na curva de calibração. O método foi aplicado em sete amostras reais postmortem e em apenas um caso foi observada uma diferença significativa (300%) entre os valores quantificados no ST periférico e central. Os antidepressivos tricíclicos AMI, NTR, IMI e DES foram avaliados no HV e o efeito matriz foi detectado para os dois últimos analitos. O método foi otimizado e validado utilizando solução salina adicionada de AMI e NTR. O limite de detecção igual a 5 ng/mL, foi obtido com a redução da voltagem da fonte de íons do espectrômetro de massa para 50 eV. Coeficientes de variação foram inferiores a 15%. Os procedimentos validados foram aplicados em seis amostras reais de HV. A relação encontrada entre os valores obtidos no ST periférico e HV foi de aproximadamente 0,1. A extração acelerada por solvente (ASE) e, posteriormente, a extração em fase sólida (SPE) foram as técnicas de separação dos analitos da matriz fígado. Ao término das citadas extrações, os antidepressivos foram analisados no GC-MS. Para esta matriz sólida, são necessários mais estudos, pois os valores encontrados nos ensaios analíticos estão em desacordo com as diretrizes utilizadas na validação dos métodos
Antidepressants belong to an important class of drugs investigated in forensic toxicology. In cases of samples from corpses, the interval between death and obtaining the biological specimens can provide the postmortem redistribution of these drugs. Aiming to elucidate this phenomenon, analytical methods were developed and applied using whole blood (WB), vitreous humor (VH) and liver. For samples of WB and HV, the extraction method chosen and validated was the three-phase liquid phase microextraction (LPME). Hollow fibers consist of polypropylene, with a length of 8 cm each were treated with dodecane organic solvent (organic phase) resulting in a membrane with selective permeability. Into the lumen of these fibers was added formic acid 0.1 mol/ L (acceptor phase). In the vial containing 3.5 mL of NaOH 0.1 mol / L (donor phase) was spiked 0.5 ml of biological fluids (WB or VH). Subsequently, the samples were injected in GC-MS without derivatization reactions. The study of the ST included antidepressants amitriptyline (AMI), nortriptyline (NTR), imipramine (IMI), desipramine (DES), clomipramine (CLO), desmethylclomipramine (DMC), fluoxetine (FLU) and norfluoxetine (NFL). The quantification limits for these substances were below the therapeutic levels (20 ng / ml). The mean coefficients of variation and separate intradays were respectively 9.7 and 9.8%. The calibration curves showed linearity between concentrations of 20 to 1200 ng / mL. The validation of the integrity of the dilution parameter assured measurement higher than the limit shown in the calibration curve quantities. The method was applied to seven real postmortem samples and in one case a significant difference (300%) between the measured values in the peripheral and central ST was observed. The tricyclic antidepressants AMI, NTR, IMI and DES were evaluated in VH and the matrix effect was detected in the last two analytes. The method was optimized and validated using saline spiked AMI and NTR. The limit of detection (5 ng/ml) was obtained by reducing the voltage of the ion source of the mass spectrometer 50 eV. Coefficients of variation were below 15%. The procedures were validated in six real samples of HV. The relationship found between the values obtained in the peripheral ST and HV was approximately 0.1. Accelerated solvent extraction (ASE) and subsequently the solid phase extraction (SPE) were the techniques of separation of analytes liver matrix. At the end of the cited extractions, antidepressants were analyzed in GC-MS. To this solid tissue, further studies are needed, because the values found in the analytical tests were not in accordance with the guidelines used in the validation of the methods
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Humans , Postmortem Changes , Biotransformation , Antidepressive Agents/analysis , Vitreous Body/pathology , Chromatography, Gas , Forensic Toxicology/instrumentation , Liquid Phase Microextraction , Forensic Medicine , Gas Chromatography-Mass Spectrometry/instrumentationABSTRACT
OBJECTIVE: To evaluate the effects of the pH condition in which the plasma samples were prepared on the matrix effects of HPLC-MS/MS analysis of stilbene compounds.
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An accurate and precise developed LCMS method was validated for the determination of pregabalin in human plasma. Validation was carried out according to US FDA guidelines. Validation data showed were within the limits. No matrix effect was found in different sources of human plasma tested. Dilution integrity, Lower limit of quantitation were also within the limit. The Mean extraction recovery of pregabalin was satisfactory.