Meeting the International Conference on Harmonisation's Guidelines on Validation of Analytical Procedures: quantification as exemplified by a near-infrared reflectance assay of paracetamol in intact tablets

This Perspective explains how the International Conference on Harmonisation's Guidelines on Validation of Analytical Procedures for quantitative methods can be met by near-infrared (NIR) assays of intact pharmaceutical products. Each of the validation characteristics (accuracy, precision, specificity, detection limit, quantification limit, linearity, range, robustness and system suitability testing) is defined, examined for their relevance to quantitative methods and examples given on how they may be used to demonstrate that near-infrared assays are fit for purpose. Methods for preparing samples for calibration are given in detail. The intention is to provide information so that a pharmaceutical manufacturer could validate a method suitable for an application for a variation of a marketing authorisation for an existing product and use a NIR assay instead of the previous method. The perspective is illustrated in detail using a NIR reflectance assay of paracetamol in intact tablets. This proven assay gives results comparable to the British Pharmacopeia ultraviolet assay for paracetamol, the standard errors of calibration and prediction for the NIR method being 0.48% w/w and 0.71% w/w respectively. The method is also precise, the standard deviation and coefficient of variation for six NIR assays on the same day being 0.14% w/w and 0.16% w/w respectively, while measurements over six consecutive days gave 0.31% w/w and 0.36% w/w respectively.     

Quantification of paracetamol in intact tablets using near-infrared transmittance spectroscopy.

Production batch samples of paracetamol tablets and specially prepared out-of-specification batches covering the range 90-110% of the stated amount (500 mg) were analysed by the BP official UV assay and by NIR transmittance spectroscopy. NIR measurements were made on 20 intact tablets from each batch, scanned five times each (10 min measurement time per batch) over the spectral range 6000-11,520 cm-1. An average spectrum was calculated for each batch. Partial least squares (PLS) regression models were set up using a calibration set (20 batches) between the NIR response and the reference tablet paracetamol content (UV). Various pre-treatments of the spectra were examined; the smallest relative standard error of prediction (0.73%) was obtained using the first derivative of the absorbance over the full spectrum. Only two principal components were required for the PLS model to give a good relationship between the spectral information and paracetamol content. Applying this model to the validation set (15 batches) gave a mean bias of -0.08% and a mean accuracy of 0.59% with relative standard deviations of 0.75 and 0.44%, respectively. The proposed method is non-destructive and therefore lends itself to on-line/at-line production control purposes. The method is easy to use and does not require a knowledge of the mass of the tablets.     

Application of transmission near-infrared spectroscopy to uniformity of content testing of intact steroid tablets

Transmission near-infrared (NIR) spectroscopy was used for the rapid and non-destructive determination of the content of a hormone steroid in single intact tablets. Tablets produced for clinical trial purposes containing 5, 10, 15, 20 and 30 mg (2.94, 5.88, 8.82, 11.76 and 17.64% m/m, respectively) were used to develop calibration models without the need to specially prepare any out of specification tablets. Reference values for the individual tablets used in the NIR calibration models and test set were measured by reversed-phase high performance liquid chromatography (HPLC). Partial least squares regression using standard normal variate transformed second-derivative spectra over the range 800 to 1040 nm gave the optimum calibration model with a standard error of calibration of 0.52 mg per tablet. Measurements of an independent test set gave comparable results (standard error of prediction 0.31 mg per tablet). Measurement errors for a single tablet (RSD < 2.5% for a given active level) were sufficiently small to allow the procedure to be applied to pharmacopoeial uniformity of content testing of batches of these tablets and permitted the non-destructive testing of 30 tablets in under 20 min as compared to 6 h by HPLC.     

Non-invasive determination of ethanol, propylene glycol and water in a multi-component pharmaceutical oral liquid by direct measurement through amber plastic bottles using Fourier transform near-infrared spectroscopy

Fourier transform near-infrared (FT-NIR) spectroscopy was used to quantify rapidly the ethanol (34-49% v/v), propylene glycol (20-35% v/v) and water (11-20% m/m) contents within a multi-component pharmaceutical oral liquid by measurement directly through the amber plastic bottle packaging. Spectra were collected in the range 7302-12,000 cm-1 and calibration models set-up using partial least-squares regression (PLSR) and multiple linear regression. Reference values for the three components were measured using capillary gas chromatography (ethanol and propylene glycol) and Karl Fischer (water) assay procedures. The calibration and test sets consisted of production as well as laboratory batches that were made to extend the concentration ranges beyond the natural production variation. The PLSR models developed gave standard errors of prediction (SEP) of 1.1% v/v for ethanol, 0.9% v/v for propylene glycol and 0.3% m/m for water. For each component the calibration model was validated in terms of: linearity, repeatability, intermediate precision and robustness. All the methods produced statistically favourable outcomes. Ten production batches independent of the calibration and test sets were also challenged against the PLSR models, giving SEP values of 1.3% v/v (ethanol), 1.0% v/v (propylene glycol) and 0.2% m/m (water). NIR transmission spectroscopy allowed all three liquid constituents to be non-invasively measured in under 1 min.     

Enantiomeric impurity determination of levetiracetam using capillary electrochromatography

CEC was used to develop a method for the enantiomeric excess determination of levetiracetam, an antiepileptic drug. Different types of calibration curve were evaluated for use in the range between 0.01 and 1 mg/mL when aniracetam was used as an internal standard. The method gave comparable results when only the areas of the impurity were used in the calibration curve. The predicted detection and quantification limits from the S/N were 1.1 and 3.6 microg/mL, respectively. However, experimental results showed that LOD and LOQ were underestimated. Repeatability of injection was demonstrated by the RSD values obtained for retention time, resolution, ratios of the areas impurity/internal standard, and areas of impurity and internal standard individually, which were below or equal to 9.30%. The between-days variability experiments indicated that it is better to make a calibration curve daily. The finally selected calibration curves were used to test the accuracy of the developed method on bulk samples and Keppra tablets containing 250 mg levetiracetam. Both selected calibration curves performed similarly. The one using the internal standard information gave overall recoveries between 88 and 118%, while the one using areas gave results between 84 and 118%.     

Measurement of the percentage volume particle size distribution of powdered microcrystalline cellulose using reflectance near-infrared spectroscopy

This is the first reported method for determining the percentage volume particle size distribution of a powder (microcrystalline cellulose) by near-infrared (NIR) reflectance spectroscopy. A total of 113 samples of powdered microcrystalline cellulose were used from six different commercially available grades, with different moisture contents (range: 0.9-4.8% m/m). NIR reflectance measurements of these samples were made in narrow soda glass vials. Reference particle size data for the samples were acquired by laser diffraction. The NIR data were then calibrated to measure particle size by partial least squares regression. The effects of a range of different NIR data pre-treatments on calibration and prediction precision were investigated. Overall, simple absorbance data were found to produce regression models with the best predictive ability (root mean square error of prediction = 0.90%). The method was also found to be insensitive to moisture content.     

废旧涤/棉混纺织物近红外定量分析模型的建立及预测

将中红外光谱筛选出的598个纯涤、纯棉及涤/棉混纺样本采用GB/T 2910.11-2009法测定其涤、棉准确含量,其中校正集样本252个,验证集样本346个。使用便携式近红外光谱仪获取样本的原始近红外光谱(NIRS)。校正集样本依据回归系数的分布趋势和范围选取最佳建模谱区,并采用差分一阶导、S-G平滑和均值中心化相结合的方法对原始光谱进行预处理,利用偏最小二乘法(PLS)建立涤/棉混纺织物中涤含量的近红外(NIR)定量分析模型。同时分析了样本颜色对NIRS的影响,探讨了斜线光谱样本、奇异样本和不同组织结构织物对模型预测效果的影响。结果表明:利用PLS法建立的涤/棉混纺织物定量分析模型最优组合包含1个光谱区间和9个主成分因子,校正集相关系数(RC)为0.998,标准偏差(SEC)为0.908。为验证所建模型的有效性和实用性,对346个未参与建模的涤棉样本进行了预测,并将预测结果与国标法测定值进行方差分析,两种方法结果无显著差异,预测正确率达97%以上。模型的建立为废旧涤/棉混纺织物快速、无损分拣提供了基础数据库。     

Active content determination of non-coated pharmaceutical pellets by near infrared spectroscopy: Method development, validation and reliability evaluation

A robust near infrared (NIR) method able to quantify the active content of pilot non-coated pharmaceutical pellets was developed. A protocol of calibration was followed, involving 2 operators, independent pilot batches of non-coated pharmaceutical pellets and two different NIR acquisition temperatures. Prediction models based on Partial Least Squares (PLS) regression were then carried out. Afterwards, the NIR method was fully validated for an active content ranging from 80 to 120% of the usual active content using new independent pilot batches to evaluate the adequacy of the method to its final purpose. Conventional criteria such as the R², the Root Mean Square Error of Calibration (RMSEC), the Root Mean Square Error of Prediction (RMSEP) and the number of PLS factors enabled the selection of models with good predictive potential. However, such criteria sometimes fail to choose the most fitted for purpose model. Therefore, a novel approach based on accuracy profiles of the validation results was used, providing a visual representation of the actual and future performances of the models. Following this approach, the prediction model using signal pre-treatment Multiplicative Scatter Correction (MSC) was chosen as it showed the best ability to quantify accurately the active content over the 80-120% active content range. The reliability of the NIR method was tested with new pilot batches of non-coated pharmaceutical pellets containing 90 and 110% of the usual active content, with blends of validation batches and industrial batches. All those batches were also analyzed by the HPLC reference method and relative errors were calculated: the results showed low relative errors in full accordance with the results obtained during the validation of the method, indicating the reliability of the NIR method and its interchangeability with the HPLC reference method.     

Development of a liquid chromatography/tandem mass spectrometry assay for the quantification of Aplidin,a novel marine-derived antineoplastic agent,in human plasma

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay was developed and validated to quantify a novel marine-derived depsipeptide, Aplidin, in human plasma. The method was validated to demonstrate the specificity, recovery, limit of quantitation (LOQ), accuracy, and precision of measurements. The calibration range for Aplidin was established using Aplidin standards from 0.05-50 ng/mL in blank human plasma. The multiple reaction monitoring, based on the transition m/z 1110.7 --> 295.3, was specific for Aplidin, and that based on the transition m/z 1112.6 --> 297.3 was specific for didemnin B (the internal standard); no endogenous materials interfered with the analysis of Aplidin and didemnin B from blank human plasma. The assay was linear over the concentration range 0.05-50.0 ng/mL. The correlation coefficients for the calibration curves ranged from 0.9979 to 0.9999. The mean intra- and interday accuracies for all calibration standards (n = 12) ranged from 97 to 106% (相似文献   

In-line monitoring of extraction process of scutellarein from Erigeron breviscapus (vant.) Hand-Mazz based on qualitative and quantitative uses of near-infrared spectroscopy

The application of near-infrared (NIR) spectroscopy for in-line monitoring of extraction process of scutellarein from Erigeron breviscapus (vant.) Hand-Mazz was investigated. For NIR measurements, two fiber optic probes designed to transmit NIR radiation through a 2 mm pathlength flow cell were utilized to collect spectra in real-time. High performance liquid chromatography (HPLC) was used as a reference method to determine scutellarein in extract solution. Partial least squares regression (PLSR) calibration model of Savitzky-Golay smoothing NIR spectra in the 5450-10,000 cm(-1) region gave satisfactory predictive results for scutellarein. The results showed that the correlation coefficients of calibration and cross validation were 0.9967 and 0.9811, respectively, and the root mean square error of calibration and cross validation were 0.044 and 0.105, respectively. Furthermore, both the moving block standard deviation (MBSD) method and conformity test were used to identify the end point of extraction process, providing real-time data and instant feedback about the extraction course. The results obtained in this study indicated that the NIR spectroscopy technique provides an efficient and environmentally friendly approach for fast determination of scutellarein and end point control of extraction process.     

Comparison of near infrared and microwave resonance sensors for at-line moisture determination in powders and tablets

In this paper we demonstrate the feasibility of replacing KF for water content testing in bulk powders and tablets with at-line near infrared (NIR) or microwave resonance (MR) methods. Accurate NIR and MR prediction models were developed with a minimalistic approach to calibration. The NIR method can accurately predict water content in bulk powders in the range of 0.5-5% w/w. Results from this method were compared to a MR method. We demonstrated excellent agreement of both NIR and MR methods for powders vs. the reference KF method. These methods are applicable to in-process control or quality control environments. One of the aims of this study was to determine if a calibration developed for a particular product could be used to predict the water content of another product (with related composition) but containing a different active pharmaceutical ingredient (API). We demonstrated that, contrary to the NIR method, a general MR method can be used to predict water content in two different types of blends. Finally, we demonstrated that a MR method can be developed for at-line moisture determination in tablets.     

Development of a liquid chromatography/tandem mass spectrometry assay for the quantification of PM02734, a novel antineoplastic agent, in dog plasma

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay was developed and validated to quantify a novel antineoplastic agent, PM02734, in dog plasma. The method was validated to demonstrate the specificity, limit of quantification (LOQ), accuracy, and precision of measurements. The calibration range for PM02734 was established using PM02734 standards from 0.05 to 100 ng/mL in blank plasma. The dominating ions were doubly charged molecular ions [M+2H]2+ at m/z 740.0 instead of singly charged ones at m/z 1478.4. The selected reaction monitoring (SRM), based on the m/z 740.0 --> 212.2 transition, was specific for PM02734, and that based on the m/z 743.8 --> 212.2 transition was specific for deuterated PM02734 (the internal standard, IS); no endogenous materials interfered with the analysis of PM02734 and IS from blank plasma. The assay was linear over the concentration range 0.05-100 ng/mL. In terms of sensitivity of assay 0.05 ng/mL is a very low LLOQ, especially considering PM02734 is a peptide. The correlation coefficients for the calibration curves ranged from 0.9990 to 0.9999. The mean intraday and interday accuracies for all calibration standards (n = 9) ranged from 93 to 111% (< or =11% bias) in dog plasma, and the mean interday precision for all calibration standards was less than 6.4%. The mean intra- and interday assay accuracy for all quality control replicates in dog plasma (n = 9), determined at each QC level throughout the validated runs, ranged from 85-111% (< or =15% bias) and from 99-109% (< or =9% bias), respectively. The mean intra- and interday assay precision was less than 12.1 and 13.3% for all QC levels, respectively. The assay has been used to support preclinical pharmacokinetic (PK) and toxicokinetic studies. The results showed that preclinical samples could be monitored for PM02734 up to 168 h after dosing, which allowed us to identify multiple elimination phases and accurately estimate PK information.     

The use of FT-NIR for API content assay in organic solvent media: a single calibration for multiple downstream processing streams

The use of near infrared spectroscopy (NIRS) in downstream solvent based processing steps of an active pharmaceutical ingredient (API) is reported. A single quantitative method was developed for API content assessment in the organic phase of a liquid–liquid extraction process and in multiple process streams of subsequent concentration and depuration steps. A new methodology based in spectra combinations and variable selection by genetic algorithm was used with an effective improvement in calibration model prediction ability. Root mean standard error of prediction (RMSEP) of 0.05 in the range of 0.20–3.00% (w/w) was achieved. With this method, it is possible to balance the calibration data set with spectra of desired concentrations, whenever acquisition of new spectra is no longer possible or improvements in model's accuracy for a specific selected range are necessary. The inclusion of artificial spectra prior to genetic algorithms use improved RMSEP by 10%. This method gave a relative RMSEP improvement of 46% compared with a standard PLS of full spectral length.     

Simultaneous measurement of glucose and glutamine in aqueous solutions by near infrared spectroscopy

A method is described for measuring the concentrations of both glucose and glutamine in binary mixtures from near infrared (NIR) absorption spectra. Spectra are collected over the range from 5000–4000/cm (2.0–2.5μm) with a 1-mm optical path length. Glucose absorbance features at 4710, 4400, and 4300/cm and glutamine features at 4700, 4580, and 4390/cm provide the analytical information required for the measurement. Multivariate calibration models are generated by using partial least squares (PLS) regression alone and PLS regression combined with a preprocessing digital Fourier filtering step. The ideal number of PLS factors and spectral range are identified separately for each analyte. In addition, the optimum Fourier filter parameters are established for both compounds. The best overall analytical performance is obtained by combining Fourier filtering and PLS regression. Glucose measurements are established over the concentration range from 1.66–59.91 mM, with a standard error of prediction (SEP) of 0.32 mM and a mean percent error of 1.84%. Glutamine can be measured over the concentration range from 1.10–30.65 mM with a SEP of 0.75 mM and a mean percent error of 6.67%. These results demonstrate the analytical utility of NIR spectroscopy for monitoring glucose and glutamine levels in mammalian and insect cell cultures.     

Reflectance near-infrared spectroscopic method with Chemometric techniques for simultaneous determination of Chondroitin, glucosamine, and methyl sulfonyl methane

Reflectance near-IR (RNIR) spectroscopy was used for the simultaneous determination of chondroitin (CH), glucosamine (GO), and methyl sulfonyl methane (MSM) in tablets. Simple sample preparation was done by grinding, sieving, and compression of the tablets for improving RNIR spectra. Principal component regression and partial least squares (PLS-1 and PLS-2) were successfully applied to quantify the three components in the studied mixture using information included in RNIR spectra in the range of 4350-9100 cm(-1). The calibration model was developed with drug concentration ranges of 14.5-44.2% (w/w) for CH, 18.4-55.3% (w/w) for GO, and 6-18.6% (w/w) for MSM with addition of tablet excipients to the calibration set in the same ratio as in the tested tablets. The calibration models were evaluated by internal validation, cross-validation, and external validation using synthetic and pharmaceutical preparations. The proposed method was applied for analysis of six batches of the pharmaceutical product. The results of the proposed method were compared with the results of the pharmacopoeial method for the same batch of the pharmaceutical product. No significant differences between the results were found. The RNIR method is accurate and precise, and can be used for QC of pharmaceutical products.     

Influence of data pre-processing on the quantitative determination of the ash content and lipids in roasted coffee by near infrared spectroscopy

In near-infrared (NIR) measurements, some physical features of the sample can be responsible for effects like light scattering, which lead to systematic variations unrelated to the studied responses. These errors can disturb the robustness and reliability of multivariate calibration models. Several mathematical treatments are usually applied to remove systematic noise in data, being the most common derivation, standard normal variate (SNV) and multiplicative scatter correction (MSC). New mathematical treatments, such as orthogonal signal correction (OSC) and direct orthogonal signal correction (DOSC), have been developed to minimize the variability unrelated to the response in spectral data. In this work, these two new pre-processing methods were applied to a set of roasted coffee NIR spectra. A separate calibration model was developed to quantify the ash content and lipids in roasted coffee samples by PLS regression. The results provided by these correction methods were compared to those obtained with the original data and the data corrected by derivation, SNV and MSC. For both responses, OSC and DOSC treatments gave PLS calibration models with improved prediction abilities (4.9 and 3.3% RMSEP with corrected data versus 7.1 and 8.3% RMSEP with original data, respectively).     

Sensitive HPLC-ESI-MS method for the determination of tiotropium in human plasma

A sensitive high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) assay is established for the determination of tiotropium in human plasma using benzyltriethylammonium chloride as the internal standard (IS). After being treated with C(18) cartridges, plasma samples are separated by HPLC on a reversed-phase C(18) column with a mobile phase of 40mM ammonium acetate buffer-methanol (56:44, v/v). Tiotropium is determined in a single-quadrupole MS. HPLC-ESI-MS is performed in the selected ion monitoring mode using target ions at m/z 392.0 for tiotropium and m/z 192.3 for the IS. The calibration curve is linear over the range 1.5-30 pg/mL. The intra- and inter-assay variability values are less than 10.1% and 13.6%, respectively. The mean plasma extraction recovery of tiotropium is 92.3 +/- 5.0%. The method has been successfully applied to studying the pharmacokinetics of tiotropium in healthy Chinese volunteers.     

Electrochemical studies and differential pulse polarographic analysis of lansoprazole in pharmaceuticals

The electrochemical reduction of lansoprazole was investigated by cyclic voltammetry and direct current and differential pulse polarography. The reduction potential was -1.32 V vs. Ag/AgCl with a dropping mercury electrode in a supporting electrolyte consisting of phosphate buffer (pH 9.0)-tetramethylammonium iodide (4 + 1). The reversibility of the electrode reaction and the type of limiting current were studied. The temperature coefficient and the diffusion constant were determined. A mechanism for the electrode reaction was proposed. A new simple and sensitive differential pulse polarographic method was also developed for the quantification of lansoprazole. A linear calibration graph was obtained in the range 0.04-11.35 micrograms ml-1. The limit of detection was 0.03 microgram ml-1 and the intra- and inter-day precisions were 0.84-2.32 and 0.72-3.09%, respectively. The developed method was applied to six different commercial pharmaceutical capsule preparations containing enteric-coated granules. The relative standard deviations ranged from 1.36 to 2.85%. Recovery studies for the accuracy of the method were performed by adding a synthetic mixture to known amounts of lansoprazole and the mean recovery was 100.45%. The data obtained from commercial preparations were compared with those from a published spectrophotometric method. No difference was found statistically.     

超高效液相色谱-质谱/质谱联用法测定人血浆中的辛伐他汀

建立了超高效液相色谱-质谱/质谱联用法(UPLC-MS/MS)测定人血浆中辛伐他汀的浓度。血浆样品经乙醚-正己烷-异丙醇(体积比为80∶20∶3)提取,以洛伐他汀为内标,采用ACQUITY UPLCTM BEH C18柱(50 mm×2.1 mm,1.7 μm)分离,以乙腈-10 mmol/L乙酸铵水溶液(体积比为85∶15)为流动相,流速为0.25 mL/min,通过电喷雾离子化,采用多反应监测(MRM)方式进行正离子检测。线性范围为0.051～20.4 ng/mL,日内及日间测定的相对标准偏差不高于10%,平均回收率为91.6%。方法灵敏度高,分析速度快,操作简便,适用于辛伐他汀药物动力学和生物等效性研究。     

Simultaneous determination of six components in commercial Roukou Wuwei pills using ultra‐high‐performance liquid chromatography with diode‐array detector

An efficient ultra‐performance liquid chromatography with diode‐array detector method was established for simultaneous determination of six active components in Roukou Wuwei pills, namely gallic acid, piperine, costundide, dehydrocostus lactone, isoalantolactone and alantolactone. Chromatographic separation of six components was successfully achieved on an Waters BEH C₁₈ column (50 × 2.1 mm, 1.7 μm) with a mobile phase composed of acetonitrile and water using a gradient elution. Gallic acid and piperine were detected at 270 nm and 343 nm, respectively; while costundide, dehydrocostus lactone, isoalantolactone and alantolactone were simultaneously measured at 225 nm. All six calibration curves showed good linearity (R² ≥ 0.9994) between the peak area of each component and corresponding concentration. Relative standard deviations for inter‐ and intra‐day precisions were <0.45 and 0.77%, respectively. The mean recovery rates ranged from 96.72 to 102.2% with relative standard deviations <2.07%. The developed method was validated in terms of linearity, precision and accuracy and then successfully applied for the quality control of commercial Roukou Wuwei samples.