Automation of the Whole-Blood Thiopurine S-Methyltransferase (TPMT) Phenotyping Assay Using the Biomek NXP and Biomek i5 Liquid-Handling Workstations

2021 ◽  
pp. 247263032110088
Author(s):  
Rachel L. Griffiths ◽  
Jonathan D. Berg
Keyword(s):  
Lower Limit ◽  
High Performance ◽  
Enzyme Assay ◽  
Liquid Handling ◽  
Automated Method ◽  
Coefficients Of Variation ◽  
Primary Sample ◽  
Limit Of Quantitation ◽  
Assay Precision ◽  
Automated Methods

Assessment of thiopurine S-methyltransferase (TPMT) status is required before commencing thiopurine treatment to reduce the potential for adverse drug reactions. Our laboratory has provided a national TPMT phenotyping service since 2003. Our assay uses 6-thioguanine as substrate and detection of 6-methylthioguanine via high-performance liquid chromatography (HPLC) fluorescence. Here, we report the automation of this complex, labor-intensive, manual assay using the Biomek NXP and Biomek i5 liquid-handling workstations. We optimized assay performance and validated for precision, linearity, and lower limit of quantitation. We also compared results from the manual and automated methods. Primary sample mixing and aliquoting were performed on the Biomek NXP. On-board inversions ( n = 10) replaced offline mixing. No carryover was observed. Eleven percent of primary sample tubes were incompatible with the Biomek NXP, and these were assayed manually. The Biomek i5 was used to automate the enzyme assay. Optimum vortex mixing was achieved at 2500 rpm for 60 s, and the temperature was set to 37.0 °C for the 60 min enzyme incubation. Intra- and inter-assay precision were excellent, with coefficients of variation (CVs) of ≤2.3% and ≤7.4%, respectively, for patient samples. Linearity was demonstrated up to 199 mIU/L ( R2 = 0.992), with a lower limit of quantitation of 3.9 mIU/L. Correlation between the manual and automated methods was good ( R2 = 0.979, n = 405), with results being interchangeable. We have successfully developed and validated a novel automated method for the TPMT phenotyping enzyme assay. The two methods are cost-neutral. Automation of other complex enzyme assays may be possible using this approach.

Analysis of 8-methoxypsoralen by high-performance liquid chromatography

1990 ◽  
Vol 36 (11) ◽  
pp. 1956-1957 ◽  
Author(s):  
C H Ketchum ◽  
C A Robinson ◽  
S T Huang
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Lower Limit ◽  
High Performance ◽  
Limit Of Detection ◽  
Extraction Recovery ◽  
Rapid Procedure ◽  
Standard Curve ◽  
Assay Precision ◽  
Interassay Precision

Abstract We report a simple and rapid procedure for assaying 8-methoxypsoralen (8-MOP) in plasma by high-performance liquid chromatography (HPLC). The standard curve for the assay is linear for 8-MOP from 15 to 500 micrograms/L (y = 0.002x-0.01, r = 0.99) with a lower limit of detection of 1.5 micrograms/L. Intra-assay precision (CV) was 6.0% at the 100 micrograms/L concentration and 10.0% at 50 micrograms/L (n = 30 each). Interassay precision was 6.4% at 100 micrograms/L and 7.0% at 50 micrograms/L (n = 50 each). Extraction recovery of 8-MOP was 98%. Common antiarrhythmics, sedatives, and hypnotics were found not to interfere.

scholarly journals Determination of Tianeptine in Tablets by High-Performance Liquid Chromatography with Fluorescence Detection

2007 ◽  
Vol 90 (3) ◽  
pp. 720-724
Author(s):  
Sevgi Tatar Ulu
Keyword(s):  
Lower Limit ◽  
High Performance ◽  
Limit Of Detection ◽  
Internal Standard ◽  
Linear Calibration ◽  
Fluorescence Detector ◽  
Relative Standard ◽  
Validation Parameters ◽  
Limit Of Quantitation

Abstract A sensitive and selective high-performance liquid chromatographic method has been developed for the determination of tianeptine (Tia) in tablets. The method is based on derivatization of Tia with 4-chloro-7-nitrobenzofurazan (NBD-Cl). A mobile phase consisting of acetonitrile10 mM orthophosphoric acid (pH 2.5; 77 + 23) was used at a flow rate of 1 mL/min on a C18 column. The Tia-NBD derivative was monitored using a fluorescence detector, with emission set at 520 nm and excitation at 458 nm. Gabapentin was selected as an internal standard. Linear calibration graphs were obtained in the concentration range of 45300 ng/mL. The lower limit of detection (LOD) was 10 ng/mL at a signal-to-noise ratio of 4. The lower limit of quantitation (LOQ) was 45 ng/mL. The relative standard values for intra- and interday precision were <0.46 and <0.57%, respectively. The recovery of the drug samples ranged between 98.89 and 99.85%. No chromatographic interference from the tablet excipients was found. The proposed method was validated in terms of precision, robustness, recovery, LOD, and LOQ. All the validation parameters were within the acceptance range. The proposed method was applied for the determination of Tia in commercially available tablets. The results were compared with those obtained by an ultraviolet spectrophotometric method using t- and F-tests.

Measurement of T-2 and HT-2 Toxins in Eggs by High-Performance Liquid Chromatography with Fluorescence Detection†

2006 ◽  
Vol 69 (11) ◽  
pp. 2773-2776 ◽  
Author(s):  
CHRIS M. MARAGOS
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Fluorescence Detection ◽  
High Performance ◽  
Limit Of Detection ◽  
Immunoaffinity Column ◽  
Florisil Column ◽  
Coefficients Of Variation ◽  
Limit Of Quantitation ◽  
Liquid Chromatographic

T-2 toxin is a mycotoxin produced by several species of common fungi capable of infesting human food and animal feeds. Lower-quality feeds given to chickens may be contaminated with T-2 toxin, which may affect their health. The literature suggests that T-2 toxin is transmitted from the hen to the eggs. This article describes the development of a liquid chromatographic assay for T-2 and the related mycotoxin HT-2 in eggs. T-2 and HT-2 toxins were isolated from spiked eggs with a tandem charcoal-alumina-Florisil column and immunoaffinity column cleanup. The isolated toxins were derivatized with the fluorophore 1-anthroyl nitrile, separated by high-performance liquid chromatography, and quantitated by fluorescence. The limit of detection of the method was 1 ng ml−1 (parts per billion) of T-2 and HT-2 in whole (with shell removed) eggs. The limit of quantitation for both toxins was 5 ng ml−1. Recoveries from spiked eggs over the range from 5 to 50 ng ml−1 averaged 89.2% for T-2 and 100.3% for HT-2, with coefficients of variation of 3.5 and 8.2%, respectively. This method is sensitive enough to be used to check for the presence of T-2 or HT-2 toxins in eggs.

scholarly journals Development and Validation of RP-HPLC Method for Ziprasidone Hydrochloride Monohydrate

2016 ◽  
Vol 8 (02) ◽  
Author(s):  
Abhay R. Shirode ◽  
Arpita Nath ◽  
Vilasrao Kadam
Keyword(s):  
Lower Limit ◽  
High Performance ◽  
Limit Of Detection ◽  
High Performance Liquid Chromatographic ◽  
Hplc Method ◽  
Rp Hplc ◽  
Relative Standard ◽  
Limit Of Quantitation ◽  
Water Ph ◽  
Hydrochloride Monohydrate

A new isocratic simple and rapid reverse phase high performance liquid chromatographic (RP-HPLC) method was developed and successively validated for the estimation of ziprasidone hydrochloride monohydrate (ZHM). In this newly developed method chromatographic separation of ZHM was achieved on a Hemochrom-Intertsil C18-5U column (250 × 4.6) mm within a short runtime of 6.5min using mobile phase containing HPLC grade water (pH adjusted to 3.0 with glacial acetic acid AR) and methanol in the ratio of 45:55% v/v. ZHM was estimated with UV detection at 317nm and it was found to be eluted at 4.8min. The above mentioned method was validated as per International Conference on Harmonization (ICH) guidelines with respect to accuracy, precision, linearity, lower limit of detection (LOD) and lower limit of quantitation (LOQ) and robustness. The method was found specific for ZHM and linear (r2 =0.998) over concentrations ranging from 2 to12μg/ml. The method was found statically accurate (mean recovery = 100.46%), precise with both intra-day and inter-day relative standard deviation (RSD) values less than 1.0% and robust. The obtained results concluded that the proposed RP-HPLC method is convenient, reliable and useful in routine analysis for estimation of ZHM in its bulk form and dosage form.

Quantification of Vitamin A in Fortified Rapeseed, Groundnut and Soya Oils Using a Simple Portable Device: Comparison to High Performance Liquid Chromatography

2013 ◽  
Vol 83 (2) ◽  
pp. 122-128 ◽  
Author(s):  
Cécile Renaud ◽  
Jacques Berger ◽  
Arnaud Laillou ◽  
Sylvie Avallone
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Vitamin A ◽  
High Performance ◽  
Vitamin A Deficiency ◽  
Developed Countries ◽  
Portable Device ◽  
Least Developed Countries ◽  
Control Food ◽  
Assay Precision

Vitamin A deficiency is still one of the major public health problems in least developed countries. Fortification of vegetable oils is a strategy implemented worldwide to prevent this deficiency. For a fortification program to be effective, regular monitoring is necessary to control food quality in the producing units. The reference methods for vitamin A quantification are expensive and time-consuming. A rapid method should be useful for regular assessment of vitamin A in the oil industry. A portable device was compared to high-performance liquid chromatography (HPLC) for three plant oils (rapeseed, groundnut, and soya). The device presented a good linearity from 3 to 30 mg retinol equivalents per kg (mg RE.kg- 1). Its limits of detection and quantification were 3 mg RE.kg- 1 for groundnut and rapeseed oils and 4 mg RE.kg- 1 for soya oil. The intra-assay precision ranged from 1.48 % to 3.98 %, considered satisfactory. Accuracy estimated by the root mean squares error ranged from 3.99 to 5.49 and revealed a lower precision than HPLC (0.4 to 2.25). Although it offers less precision than HPLC, the device estimates quickly the vitamin A content of the tested oils from 3 or 4 to 15 mg RE.kg- 1.

Validated Stability Indicating HPTLC Method for the Estimation of Amoxapine in Different Stress Conditions

2019 ◽  
Vol 15 (3) ◽  
pp. 273-279
Author(s):  
Shweta G. Rangari ◽  
Nishikant A. Raut ◽  
Pradip W. Dhore
Keyword(s):  
High Performance ◽  
Limit Of Detection ◽  
Degradation Products ◽  
Analysis Data ◽  
Peak Height ◽  
Good Resolution ◽  
Stability Indicating ◽  
Limit Of Quantitation ◽  
Wide Range ◽  
Hptlc Method

Background:The unstable and/or toxic degradation products may form due to degradation of drug which results into loss of therapeutic activity and lead to life threatening condition. Hence, it is important to establish the stability characteristics of drug in various conditions such as in temperature, light, oxidising agent and susceptibility across a wide range of pH values.Introduction:The aim of the proposed study was to develop simple, sensitive and economic stability indicating high performance thin layer chromatography (HPTLC) method for the quantification of Amoxapine in the presence of degradation products.Methods:Amoxapine and its degraded products were separated on precoated silica gel 60F254 TLC plates by using mobile phase comprising of methanol: toluene: ammonium acetate (6:3:1, v/v/v). The densitometric evaluation was carried out at 320 nm in reflectance/absorbance mode. The degradation products obtained as per ICH guidelines under acidic, basic and oxidative conditions have different Rf values 0.12, 0.26 and 0.6 indicating good resolution from each other and pure drug with Rf: 0.47. Amoxapine was found to be stable under neutral, thermal and photo conditions.Results:The method was validated as per ICH Q2 (R1) guidelines in terms of accuracy, precision, ruggedness, robustness and linearity. A good linear relationship between concentration and response (peak area and peak height) over the range of 80 ng/spot to 720 ng/spot was observed from regression analysis data showing correlation coefficient 0.991 and 0.994 for area and height, respectively. The limit of detection (LOD) and limit of quantitation (LOQ) for area were found to be 1.176 ng/mL and 3.565 ng/mL, whereas for height, 50.063 ng/mL and 151.707 ng/mL respectively.Conclusion:The statistical analysis confirmed the accuracy, precision and selectivity of the proposed method which can be effectively used for the analysis of amoxapine in the presence of degradation products.

Feasibility and Evaluation of Automated Methods for Radiolabeling of Radiopharmaceutical Kits with Gallium-68

2019 ◽  
Vol 12 (3) ◽  
pp. 229-237 ◽  
Author(s):  
Alban Revy ◽  
François Hallouard ◽  
Sandrine Joyeux-Klamber ◽  
Andrea Skanjeti ◽  
Catherine Rioufol ◽  
...  
Keyword(s):  
The European Union ◽  
Preparation Time ◽  
Limit Of Quantification ◽  
Manual Method ◽  
Personnel Costs ◽  
Automated Method ◽  
Gallium 68 ◽  
The Right ◽  
First Time ◽  
Automated Methods

Objective: Recent gallium-68 labeled peptides are of increasing interest in PET imaging in nuclear medicine. Somakit TOC® is a radiopharmaceutical kit registered in the European Union for the preparation of [68Ga]Ga-DOTA-TOC used for the diagnosis of neuroendocrine tumors. Development of a labeling process using a synthesizer is particularly interesting for the quality and reproducibility of the final product although only manual processes are described in the Summary of Product (SmPC) of the registered product. The aim of the present study was therefore to evaluate the feasibility and value of using an automated synthesizer for the preparation of [68Ga]Ga-DOTA-TOC according to the SmPC of the Somakit TOC®. Methods: Three methods of preparation were compared; each followed the SmPC of the Somakit TOC®. Over time, overheads, and overexposure were evaluated for each method. Results: Mean±SD preparation time was 26.2±0.3 minutes for the manual method, 28±0.5 minutes for the semi-automated, and 40.3±0.2 minutes for the automated method. Overcost of the semi-automated method is 0.25€ per preparation for consumables and from 0.58€ to 0.92€ for personnel costs according to the operator (respectively, technician or pharmacist). For the automated method, overcost is 70€ for consumables and from 4.06€ to 6.44€ for personnel. For the manual method, extremity exposure was 0.425mSv for the right finger, and 0.350mSv for the left finger; for both the semi-automated and automated method extremity exposure were below the limit of quantification. Conclusion: The present study reports for the first time both the feasibility of using a [68Ga]- radiopharmaceutical kit with a synthesizer and the limits for the development of a fully automated process.

scholarly journals PERFORMANCE CHARACTERISTICS OF A FRET-BASED IMMUNOASSAY FOR QUANTITATION OF INFLIXIMAB ON A POINT-OF-CARE INSTRUMENT SYSTEM

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S57-S57
Author(s):  
Edgar Ong ◽  
Ruo Huang ◽  
Richard Kirkland ◽  
Michael Hale ◽  
Larry Mimms
Keyword(s):  
Whole Blood ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Quantitative Detection ◽  
Therapeutic Drug ◽  
Analytical Sensitivity ◽  
Sample Type ◽  
Hook Effect ◽  
Limit Of Quantitation ◽  
Assay Precision

Abstract Introduction A fast (<5 min), time-resolved fluorescence resonance energy transfer (FRET)-based immunoassay was developed for the quantitative detection of infliximab (IFX) and biosimilars for use in therapeutic drug monitoring using only 20 µL of fingerstick whole blood or serum at the point-of-care. The Procise IFX assay and ProciseDx analyzer are CE-marked. Studies were performed to characterize analytical performance of the Procise IFX assay on the ProciseDx analyzer. Methods Analytical testing was performed by spiking known amounts of IFX into negative serum and whole blood specimens. Analytical sensitivity was determined using limiting concentrations of IFX. Linearity was determined by testing IFX across the assay range. Hook effect was assessed at IFX concentrations beyond levels expected to be found within a patient. Testing of assay precision, cross-reactivity and potential interfering substances, and biosimilars was performed. The Procise IFX assay was also compared head-to-head with another CE-marked assay: LISA-TRACKER infliximab ELISA test (Theradiag, France). The accuracy of the Procise IFX assay is established through calibrators and controls traceable to the WHO 1st International Standard for Infliximab (NIBSC code: 16/170). Results The Procise IFX assay shows a Limit of Blank, Limit of Detection, and Lower Limit of Quantitation (LLoQ) of 0.1, 0.2, and 1.1 µg/mL in serum and 0.6, 1.1, and 1.7 µg/mL in whole blood, respectively. The linear assay range was determined to be 1.7 to 77.2 µg/mL in serum and whole blood. No hook effect was observed at an IFX concentration of 200 µg/mL as the value reported as “>ULoQ”. Assay precision testing across 20 days with multiple runs and reagent lots showed an intra-assay coefficient of variation (CV) of 2.7%, an inter-assay CV of <2%, and a total CV of 3.4%. The presence of potentially interfering/cross-reacting substances showed minimal impact on assay specificity with %bias within ±8% of control. Testing of biosimilars (infliximab-dyyb and infliximab-abda) showed good recovery. A good correlation to the Theradiag infliximab ELISA was obtained for both serum (slope=1.01; r=0.99) and whole blood (slope=1.01; r=0.98) samples (Figure 1). Conclusion Results indicate that the Procise IFX assay is sensitive, specific, and precise yielding results within 5 minutes from both whole blood and serum without the operator needing to specify sample type. Additionally, it shows very good correlation to a comparator assay that takes several hours and sample manipulation to yield results. This makes the Procise IFX assay ideal for obtaining fast and accurate IFX quantitation, thus allowing for immediate drug level dosing decisions to be made by the physician during patient treatment.

scholarly journals Development and Validation of 2-Azaspiro [4,5] Decan-3-One (Impurity A) in Gabapentin Determination Method Using qNMR Spectroscopy

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1656
Author(s):  
Nataliya E. Kuz’mina ◽  
Sergey V. Moiseev ◽  
Mikhail D. Khorolskiy ◽  
Anna I. Lutceva
Keyword(s):  
Test Procedure ◽  
Active Pharmaceutical Ingredient ◽  
Test Methods ◽  
Intermediate Precision ◽  
Coefficients Of Variation ◽  
Validation Parameters ◽  
Limit Of Quantitation ◽  
Student’S T ◽  
Development And Validation ◽  
Student’S T Test

The authors developed a 1H qNMR test procedure for identification and quantification of impurity A present in gabapentin active pharmaceutical ingredient (API) and gabapentin products. The validation studies helped to determine the limit of quantitation and assess linearity, accuracy, repeatability, intermediate precision, specificity, and robustness of the procedure. Spike-and-recovery assays were used to calculate standard deviations, coefficients of variation, confidence intervals, bias, Fisher’s F test, and Student’s t-test for assay results. The obtained statistical values satisfy the acceptance criteria for the validation parameters. The authors compared the results of impurity A quantification in gabapentin APIs and capsules by using the 1H qNMR and HPLC test methods.

scholarly journals Development of One-Step Non-Solvent Extraction and Sensitive UHPLC-MS/MS Method for Assessment of N-(n-Butyl) Thiophosphoric Triamide (NBPT) and N-(n-Butyl) Phosphoric Triamide (NBPTo) in Milk

Molecules ◽  
2021 ◽  
Vol 26 (10) ◽  
pp. 2890
Author(s):  
Chikere G. Nkwonta ◽  
Macdara O’Neill ◽  
Niharika Rahman ◽  
Mary Moloney ◽  
Patrick J. Forrestal ◽  
...  
Keyword(s):  
Field Study ◽  
High Performance ◽  
Storage Temperature ◽  
Nitrous Oxide Emissions ◽  
Milk Samples ◽  
C Storage ◽  
Fortified Milk ◽  
Limit Of Quantitation ◽  
Improved Stability ◽  
Sensitivity Specificity

N-(n-butyl) thiophosphoric triamide (NBPT) is a urease inhibitor utilised in urea-based fertilizers. In Ireland, fertilizer treated with NBPT is applied to pasture to mitigate both ammonia and nitrous oxide emissions, but concerns arise as to the potential for residues in milk products. A quick ultrafiltration extraction and ultra-high performance liquid chromatography coupled with mass spectrometry triple quadrupole (UHPLC-MS/MS) quantitation method was developed and validated in this study. The method was applied in the analysis of samples collected from a field study investigating potential transfer of NBPT residues into milk. NBPT and NBPTo residues, were extracted from fortified milk samples and analysed on a UHPLC-MS/MS with recoveries ranging from 74 to 114%. Validation of the UHPLC-MS/MS method at low (0.0020 mg kg−1) and high (0.0250 mg kg−1) concentration levels in line with SANTE/12682/2019 showed overall trueness in the range of 99 to 104% and precision between 1 and 10%, RSD for both compounds. The limit of quantitation (LOQ) was 0.0020 mg kg−1 and other tested parameters (linearity, sensitivity, specificity, matrix effect, robustness, etc.) satisfied acceptance criteria. Stability assessment using spiked samples revealed the compounds were stable in raw and pasteurised milk for 4 weeks at –80 °C storage temperature. Maintaining samples at pH 8.5–9.0 further improved stability. Analysis of 516 milk samples from the field study found that NBPT and NBPTo concentrations were below the LOQ of 0.0020 mg kg−1, thus suggesting very low risk of residues occurring in the milk. The method developed is quick, robust, and sensitive. The method is deemed fit-for-purpose for the simultaneous determination of NBPT and NBPTo in milk.

Sign in / Sign up

Export Citation Format

Share Document