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Zhang Yun Jing Gender Reassignment

Abstract

Climate change is a significant and emerging threat to human health, especially where infectious diseases are involved. Because of the complex interactions between climate variables and infectious disease components (i.e., pathogen, host and transmission environment), systematically and quantitatively screening for infectious diseases that are sensitive to climate change is still a challenge. To address this challenge, we propose a new statistical indicator, Relative Sensitivity, to identify the difference between the sensitivity of the infectious disease to climate variables for two different climate statuses (i.e., historical climate and present climate) in non-exposure and exposure groups. The case study in Anhui Province, China has demonstrated the effectiveness of this Relative Sensitivity indicator. The application results indicate significant sensitivity of many epidemic infectious diseases to climate change in the form of changing climatic variables, such as temperature, precipitation and absolute humidity. As novel evidence, this research shows that absolute humidity has a critical influence on many observed infectious diseases in Anhui Province, including dysentery, hand, foot and mouth disease, hepatitis A, hemorrhagic fever, typhoid fever, malaria, meningitis, influenza and schistosomiasis. Moreover, some infectious diseases are more sensitive to climate change in rural areas than in urban areas. This insight provides guidance for future health inputs that consider spatial variability in response to climate change. View Full-Text

Keywords: climate change; infectious diseases; climate change-sensitive diseases; relative sensitivity; screening methodclimate change; infectious diseases; climate change-sensitive diseases; relative sensitivity; screening method

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MDPI and ACS Style

Wang, Y.; Rao, Y.; Wu, X.; Zhao, H.; Chen, J. A Method for Screening Climate Change-Sensitive Infectious Diseases. Int. J. Environ. Res. Public Health2015, 12, 767-783.

AMA Style

Wang Y, Rao Y, Wu X, Zhao H, Chen J. A Method for Screening Climate Change-Sensitive Infectious Diseases. International Journal of Environmental Research and Public Health. 2015; 12(1):767-783.

Chicago/Turabian Style

Wang, Yunjing; Rao, Yuhan; Wu, Xiaoxu; Zhao, Hainan; Chen, Jin. 2015. "A Method for Screening Climate Change-Sensitive Infectious Diseases." Int. J. Environ. Res. Public Health 12, no. 1: 767-783.

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Abstract

A specific and sensitive liquid chromatography–electrospray ionization–tandem mass spectrometric method was developed for the quantification of imatinib and its primary metabolite N-desmethyl imatinib in human plasma. Protein precipitation with methanol was used for sample preparation. High-performance liquid chromatographic separation was performed on a Thermo BDS Hypersil C18 column (4.6 × 100 mm, 2.4 µm) with methanol–water (55:45, v/v) containing 0.1% formic acid and 0.2% ammonium acetate as the mobile phase, using isocratic elution at a flow rate of 0.7 mL/min. Detection was conducted with positive electrospray ionization multiple reaction monitoring of the ion transitions at m/z 494 → 394 for imatinib, 480 → 394 for N-desmethyl imatinib and 297 → 110 for the internal standard (palonosetron). The assay was validated in the concentration ranges of 8–5,000 ng/mL for imatinib and 3–700 ng/mL for N-desmethyl imatinib. The quantification limits for imatinib and N-desmethyl imatinib were 8 and 3 ng/mL, respectively. The intra-day and inter-day precision values of the assay (expressed as percentage relative standard deviation) were less than 15% at all concentration levels within the tested range, and the accuracy values were between 85 and 115%. The established method was successfully applied to the pharmacokinetic study of imatinib mesylate capsules in 24 healthy Chinese volunteers.

Introduction

Chronic myelogenous leukemia is a myeloproliferative disorder associated with an abnormal BCR-ABL tyrosine kinase. Imatinib, a synthetic phenylaminopyrimidine derivative, which inhibits the tyrosine kinase with high selectivity, has been established as a highly effective therapy for chronic myelogenous leukemia (1) and gastrointestinal stromal tumors (2).

Imatinib is predominantly metabolized by CYP3A4 to N-desmethyl imatinib, which shows comparable biological activity to the parent drug (3). The activity of CYP3A4 displays large inter-individual variability. Therefore, a given dose of imatinib can yield very different circulating concentrations of the parent drug and its metabolites (4). Studies show that an adequate plasma concentration of imatinib is important for a good clinical response (5–6), which emphasizes the significance of therapeutic drug monitoring and pharmacokinetics investigation of imatinib.

The quantitation of plasma levels of imatinib and its primary metabolite is the key aspect of the pharmacokinetic study. Until now, the simultaneous quantification of imatinib and N-desmethyl imatinib in human plasma have been accomplished by liquid chromatography–ultraviolet detection (LC–UV) (7–14), LC–mass spectrometry (MS) (15–16) or tandem MS (MS-MS) (17–18) methods. The most frequently used method is LC–UV. However, UV detection suffers from a lack of sensitivity and a limited linear range. LC–MS-MS is considered to be the preferred method for the analysis of imatinib and N-desmethyl imatinib in complex biological samples because of its sensitivity and selectivity. However, some published works have involved complex gradient LC methods (15–18) to obtain good peak shapes and sensitivity, but a longer run time was required due to column re-equilibration. The most recent work (19) reported a gradient elution for the simultaneous determination of imatinib and N-desmethyl imatinib with a re-equilibration time of 3 min and a total run time of 6.0 min; the calibration ranges were from 10 to 2,000 ng/mL and analyte concentrations above the upper limit of quantification (ULOQ) had to be diluted and re-assayed.

Isocratic elution often provides stable baselines and constant LC–MS ionization efficiency for the analytes and requires no time-consuming preconditioning between individual runs. These are important aspects for high throughput bioassays. In this paper, a selective and robust method was developed for the simultaneous determination of imatinib and N-desmethyl imatinib by LC–MS-MS with higher sensitivity and wider linear range through the use of more straightforward isocratic elution using only a 3.8 min runtime The method showed good retention time reproducibility; therefore, it will be easy to transfer between laboratories and highly advantageous for pharmacokinetic studies. The feasibility of the proposed method has been demonstrated by the successful application to a pharmacokinetic study of imatinib mesylate capsules in healthy Chinese volunteers.

Experimental

Chemicals and reagents

Reference standards of imatinib mesylate (purity > 99.8%), N-desmethyl imatinib (purity > 99.8%), and the internal standard (IS) palonosetron hydrochloride (purity > 99.8%) were provided by Jiangsu Chia-Tai Tianqing Pharmacy Co. (Nanjing, China). Methanol [high-performance liquid chromatography (HPLC) grade] was supplied by Tedia Company (Fairfield, OH). All other chemicals and reagents were of analytical grade. All aqueous solutions were prepared with purified water (18.3 MΩ cm; Millipore, Billerica, MA).

Methods

Instrumentation and conditions

The LC–MS-MS system consisted of a Waters 2695 HPLC system (Waters, Milford, MA) with a quaternary gradient pump, an online vacuum degasser, a column oven and an autosampler, coupled to a Micromass Quattro micro triple-quadrupole mass spectrometer (Micromass, Manchester, UK) equipped with an electrospray ionization (ESI) interface. Data acquisition was performed with Masslynx 4.0 software (Micromass).

HPLC separation was performed on a Thermo BDS Hypersil C18 column (4.6 × 100 mm, 2.4 µm) maintained at 40°C with a mobile phase of methanol–water containing 0.1% formic acid and 0.2% ammonium acetate (55:45, v/v), which was delivered at 0.7 mL/min; 30% of the eluent was split into the inlet of the mass spectrometer for detection. A divert valve was used to divert the eluent to waste from 0 to 2.4 min. The autosampler was set at 4°C.

The mass spectrometer was operated in the positive ESI mode with the spray voltage set at 3 kV, nitrogen gas desolvation flow of 500 L/h at a temperature of 350°C and a sweep gas flow of 20 L/h. Quantification was performed with multiple reaction monitoring (MRM) by using argon gas collision induced dissociation and the following ion transitions: m/z 494 → 394, 480 → 394 and 297 → 110 for imatinib, N-desmethyl imatinib and palonosetron (IS), respectively, with the cone voltages all set at 30 V and the collision energy at 28 eV. Figure 1 shows the typical production scan spectra and the proposed patterns of fragmentation of the analytes and the IS.

Figure 1.

Product ion scan mass spectra: imatinib (A); N-desmethyl imatinib (B); palonosetron (IS) (C).

Figure 1.

Product ion scan mass spectra: imatinib (A); N-desmethyl imatinib (B); palonosetron (IS) (C).

Stock solutions

Stock solutions of imatinib and N-desmethyl imatinib at concentrations of approximately 800,000 and 90,000 ng/mL, respectively (both as the free base), were prepared in methanol–water (1:1, v/v). Working solutions of imatinib and N-desmethyl imatinib were prepared by serial dilution with the same solvent in the range from 80 to 50,000 ng/mL for imatinib and 30 to 7,000 ng/mL for N-desmethyl imatinib. The stock and working solutions of palonosetron hydrochloride (IS) were prepared similarly at 200,000 and 2,000 ng/mL as the free base. All solutions were stored under refrigeration (4°C) when not in use.

Plasma sample pretreatment

An aliquot of a 0.4 mL plasma sample was spiked with 40 µL of the IS and 40 µL of methanol–water (1:1, v/v), or 40 µL of the corresponding working standard solutions, for the preparation of the calibration plasma standards and quality control samples, followed by protein precipitation with the addition of 1.2 mL methanol and vortex-mixing for 1 min and centrifuging at 10,000 × g for 10 min at 4°C. The supernatant was transferred into an autosampler vial for LC–MS-MS analysis with an injection volume of 10 µL.

The calibration plasma standards of imatinib and N-desmethyl imatinib were prepared and analyzed separately to avoid possible cross-talk interferences, although these were not found in this study. To prepare the plasma calibration standards, an aliquot of 40 µL of each working standard solution was mixed precisely with 0.4 mL of blank plasma to produce the calibration standard in the ranges of 8.0–5000 ng/mL for imatinib and 3.0–700 ng/mL for N-desmethyl imatinib. The quality control (QC) samples were made at 16, 400 and 3,200 ng/mL for imatinib and 9, 90 and 500 ng/mL for N-desmethyl imatinib in the same way.

Method validation

The analytical method was validated for specificity, matrix effects, linearity, lower limit of quantification (LLOQ), accuracy, precision and recovery of measurements. The specificity was evaluated by comparing the chromatograms of six different batches of blank plasma with the corresponding spiked plasma to investigate the potential interferences near the retention times of either the analytes or the IS. The linearity of the method was determined by the analysis of a series of standard samples with concentrations from 8.0 to 5,000 ng/mL for imatinib and 3.0 to 700 ng/mL for N-desmethyl imatinib. The calibration curves were established through weighted linear least-squares regression of the peak area ratios (Y) of the analytes to the IS obtained against the corresponding concentrations (C, in ng/mL). Coefficients of correlation (r) were required to be 0.99 or better. The acceptance criterion for each back-calculated standard concentration above the LLOQ was ± 15% deviation from the nominal value, except at LLOQ. The LLOQ was defined as the concentration of the sample that could be quantified with less than 20% variation in precision (n = 6) and provided a signal-to-noise ratio ≥ 10; this was established by using six independent samples. The intra-batch and inter-batch accuracy and precision were determined by analysis of five replicates at three QC concentration levels. The criteria for acceptability of the data included accuracy within ± 15% deviation from the nominal values and precision within 15%. Recoveries of imatinib and N-desmethyl imatinib from plasma with protein precipitation by methanol were determined by comparing their peak areas in spiked plasma samples at three QC concentrations with those in samples prepared by spiking the blank plasma post-preparation with the same amounts of imatinib and N-desmethyl imatinib. The recovery of the IS was evaluated at 2,000 ng/mL. Matrix effects were caused by ionization competition occurring among imatinib, N-desmethyl imatinib, IS and endogenous co-eluting components. To evaluate the matrix effects, chromatographic peaks of imatinib, N-desmethyl imatinib and IS from the spiked solution after preparation were compared with those obtained by direct injection of the standard solutions prepared in the mobile phase at the QC concentrations.

The stability of the analytes was assessed by using triplicate spiked plasma samples containing imatinib and N-desmethyl imatinib at two concentration levels (30 and 1,600 ng/mL for imatinib; 18 and 350 ng/mL for N-desmethyl imatinib), which were analyzed after subjection to various storage and handling conditions over time periods that exceeded those applied to the actual study samples. The spiked stability samples were analyzed against a calibration curve that was obtained from spiked calibration standards prepared from freshly made stock solutions; the obtained concentrations were compared to the nominal concentrations. The mean concentration at each level should be within ± 15% of the nominal concentration. For freeze–thaw stability, samples were stored at −20°C for 24 h and thawed unassisted at room temperature. After complete thawing, samples were refrozen again under the same conditions. The freeze–thaw cycle was repeated three times and analysis was conducted on the third cycle. Short-term temperature stability was assessed by analyzing samples thawed at room temperature and kept at this temperature for 8 h, and the stability of the post-preparative samples kept at room temperature for 8 h was also evaluated. The stability of the post-preparative samples in the autosampler was conducted by re-analyzing processed samples kept in the autosampler at 4°C for 24 h. Long-term stability was determined by storing at −20°C for 75 days.

The stabilities of stock solutions of imatinib, N-desmethyl imatinib and IS (with an appropriate dilution, taking into consideration the linearity and measuring range of the detector) were evaluated by comparing the response of the stock solutions kept at 4°C for 50 days with that of freshly prepared solutions.

Method application

The validated method was applied for the determination of imatinib and its primary metabolite N-desmethyl imatinib in plasma samples in a pharmacokinetic study. Twenty-four healthy male Chinese volunteers were selected as subjects after clinical screening procedures. Each subject was fasted and administered a single oral dose of 400 mg of imatinib mesylate capsules. Venous blood samples of approximately 4 mL were collected into heparinized polypropylene tubes at pre-dose and 0.5, 1.0, 1.5, 2.5, 4.0, 6.0, 8.0, 12, 24, 36, 48, 72, 96 and 120 h after administration. Plasma was separated by centrifugation at 4,000 g for 5 min and stored at −20°C until analysis. The study was conducted at Xijing Hospital (Xi'an, China) in accordance with the principles of the Declaration of Helsinki after receiving approval from the independent ethics committee in the hospital. All subjects gave written consent for their participation after having been informed by the medical supervisor about the aim, course and possible risks of the study.

Results

Method validation

Under the proposed LC–MS-MS conditions, retention times for imatinib, N-desmethyl imatinib and the IS were 2.8 ± 0.1, 2.9 ± 0.1 and 2.8 ± 0.1 min, respectively, and the total run time was approximately 3.8 min. No obvious interferences from endogenous substances were observed. Typical chromatograms are shown in Figure 2 for blank plasma, plasma spiked with LLOQ standards of imatinib, N-desmethyl imatinib and IS, and plasma samples collected from a subject 4 h after dosing.

Figure 2.

Representative MRM chromatograms: blank plasma (A); blank plasma spiked with imatinib [8 ng/mL, LLOQ, retention time (tR) = 2.9 min] and the IS, palonosetron (200 ng/mL, tR = 2.8 min) (B); N-desmethyl imatinib (3 ng/mL, LLOQ, tR = 2.9 min) and the IS, palonosetron (200 ng/mL, tR = 2.8 min) (C); plasma sample of a subject 4 h after single oral administration of imatinib mesylate capsules (400 mg of imatinib free base) (D).

Figure 2.

Representative MRM chromatograms: blank plasma (A); blank plasma spiked with imatinib [8 ng/mL, LLOQ, retention time (tR) = 2.9 min] and the IS, palonosetron (200 ng/mL, tR = 2.8 min) (B); N-desmethyl imatinib (3 ng/mL, LLOQ, tR = 2.9 min) and the IS, palonosetron (200 ng/mL, tR = 2.8 min) (C); plasma sample of a subject 4 h after single oral administration of imatinib mesylate capsules (400 mg of imatinib free base) (D).

Suitable weighting factors were selected for linear regression because the F-tests and homoscedasticity tests, conducted by plotting residuals versus concentration, demonstrated the heteroscedasticity. Empirical weights of 1/Y, 1/C, 1/Y2 and 1/C2 were evaluated. The best weighting factor was chosen according to the percentage relative error (RE), which compares the regressed concentration computed from the regression equation obtained for each weighting factor with the nominal standard concentration. Results showed that the weighting factor of 1/C gave the least sum of absolute RE across the whole concentration range; thus, it was selected as the weighting factor. Good linear relationships were obtained over the ranges of 8–5,000 and 3–700 ng/mL for imatinib and N-desmethyl imatinib, respectively. Typical equations were Y = (2.188 ± 0.006028) C + (0.1794 ± 0.01397) (r = 0.9997 ± 0.0001) (n = 3) for imatinib and Y = (1.258 ± 0.01908) C + (0.01479 ± 0.001946) (r = 0.9997 ± 0.0001) (n = 3) for N-desmethyl imatinib. The accuracy observed for the mean of back-calculated concentrations for three calibration curves was within 97.26–106.3% and 90.90–106.2% for imatinib and N-desmethyl imatinib, respectively, whereas the inter-validation precision [percentage relative standard deviation (RSD)] of the back-calculated calibration standards ranged from 0.28 to 2.15% for imatinib and 0.10 to 3.28% for N-desmethyl imatinib. The accuracy, precision (RSD) and regression parameters of slope, intercept and correlation coefficient (r) calculated by weight (1/C) linear regression are summarized in Tables I and II. The LLOQ values were found to be 8 and 3 ng/mL for imatinib and N-desmethyl imatinib, respectively; these values are in agreement with the requirement in human pharmacokinetic study.

Table I

Accuracy and Precision (RSD) of Calibration Curve Data and Regression Parameters Calculated by Weighted (1/C) Linear Regression for Imatinib

STD-1 STD-2 STD-3 STD-4 STD-5 STD-6 STD-7 STD-8 STD-9 STD-10 Slope Intercept r
Nominal (ng/mL) 8.000 16.00 32.00 80.00 160.0 400.0 800.0 1,600 3,200 5,000 
Back-calculated (ng/mL) Validation 1 7.707 16.23 31.27 82.26 154.2 428.6 825.3 1,513 3,187 5,046 2.194 0.1896 0.9996 
Validation 2 7.663 16.39 31.79 81.61 154.1 423.1 816.7 1,529 3,206 5,025 2.187 0.1635 0.9998 
Validation 3 7.972 15.89 31.63 80.96 153.3 424.2 825.4 1,524 3,213 5,014 2.182 0.1852 0.9997 
Overall mean 7.781 16.17 31.56 81.61 153.9 425.3 822.5 1,522 3,202 5,028 2.188 0.1794 0.9997 
SD 0.1671 0.2528 0.2645 0.6506 0.4902 2.907 4.979 8.363 13.30 15.84 0.006028 0.01397 0.0001 
RSD (%) 2.15 1.56 0.84 0.80 0.32 0.68 0.61 0.55 0.42 0.31 0.28 7.79 
Accuracy (%) 97.26 101.1 98.64 102.0 96.16 106.3 102.8 95.12 100.1 100.6 
Bias (%) –2.74 1.07 –1.36 2.01 –3.84 6.32 2.81 –4.88 0.06 0.56 
STD-1 STD-2 STD-3 STD-4 STD-5 STD-6 STD-7 STD-8 STD-9 STD-10 Slope Intercept r
Nominal (ng/mL) 8.000 16.00 32.00 80.00 160.0 400.0 800.0 1,600 3,200 5,000 
Back-calculated (ng/mL) Validation 1 7.707 16.23 31.27 82.26 154.2 428.6 825.3 1,513 3,187 5,046 2.194 0.1896 0.9996 
Validation 2 7.663 16.39 31.79 81.61 154.1 423.1 816.7 1,529 3,206 5,025 2.187 0.1635 0.9998 
Validation 3 7.972 15.89 31.63 80.96 153.3 424.2 825.4 1,524 3,213 5,014 2.182 0.1852 0.9997 
Overall mean 7.781 16.17 31.56 81.61 153.9 425.3 822.5 1,522 3,202 5,028 2.188 0.1794 0.9997 
SD 0.1671 0.2528 0.2645 0.6506 0.4902 2.907 4.979 8.363 13.30 15.84 0.006028 0.01397 0.0001 
RSD (%) 2.15 1.56 0.84 0.80 0.32 0.68 0.61 0.55 0.42 0.31 0.28 7.79 
Accuracy (%) 97.26 101.1 98.64 102.0 96.16 106.3 102.8 95.12 100.1 100.6 
Bias (%) –2.74 1.07 –1.36 2.01 –3.84 6.32 2.81 –4.88 0.06 0.56 

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Table II

Accuracy and Precision (RSD) of Calibration Curve Data and Regression Parameters Calculated by Weighted (1/C) Linear Regression for N-Desmethyl Imatinib

STD-1 STD-2 STD-3 STD-4 STD-5 STD-6 STD-7 STD-8 STD-9 STD-10 Slope Intercept r
Nominal (ng/mL) 3.000 7.500 15.00 30.00 60.00 87.50 175.0 350.0 525.0 700.0 
Back-calculated (ng/mL) Validation 1 3.301 7.759 13.80 28.45 61.64 83.34 176.7 357.7 509.7 709.8 1.236 0.01535 0.9996 
Validation 2 3.154 7.539 13.53 31.42 61.95 83.42 176.4 357.1 509.7 707.2 1.270 0.01263 0.9997 
Validation 3 3.099 7.466 13.57 31.22 61.31 86.41 178.2 357.1 511.4 701.3 1.268 0.01640 0.9998 
Overall mean 3.184 7.588 13.64 30.36 61.64 84.39 182.2 357.3 524.8 726.3 1.258 0.01479 0.9997 
SD 0.1044 0.1527 0.1468 1.6602 0.3201 1.748 1.004 0.3404 1.021 4.376 0.01908 0.001946 0.0001 
RSD (%) 3.28 2.01 1.08 5.47 0.52 2.07 0.55 0.10 0.19 0.60 1.52 13.15 
Accuracy (%) 106.2 101.2 90.90 101.2 102.73 96.45 104.1 102.1 99.97 103.8 
Bias (%) 6.15 1.18 –9.10 1.20 2.73 –3.55 4.09 2.08 –0.03 3.75 
STD-1 STD-2 STD-3 STD-4 STD-5 STD-6 STD-7 STD-8 STD-9 STD-10 Slope Intercept r
Nominal (ng/mL) 3.000 7.500 15.00 30.00 60.00 87.50 175.0 350.0 525.0 700.0 
Back-calculated (ng/mL) Validation 1 3.301 7.759 13.80 28.45 61.64 83.34 176.7 357.7 509.7 709.8 1.236 0.01535 0.9996 
Validation 2 3.154 7.539 13.53 31.42 61.95 83.42 176.4 357.1 509.7 707.2 1.270 0.01263 0.9997 
Validation 3 3.099 7.466 13.57 31.22 61.31 86.41 178.2 357.1 511.4 701.3 1.268 0.01640 0.9998 
Overall mean 3.184 7.588 13.64 30.36 61.64 84.39 182.2 357.3 524.8 726.3 1.258 0.01479 0.9997 
SD 0.1044 0.1527 0.1468 1.6602 0.3201 1.748 1.004 0.3404 1.021 4.376 0.01908 0.001946 0.0001 
RSD (%) 3.28 2.01 1.08 5.47 0.52 2.07 0.55 0.10 0.19 0.60 1.52 13.15 
Accuracy (%) 106.2 101.2 90.90 101.2 102.73 96.45 104.1 102.1 99.97 103.8 
Bias (%) 6.15 1.18 –9.10 1.20 2.73 –3.55 4.09 2.08 –0.03 3.75 

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The intra-batch and inter-batch precision values were ≤8.3% for imatinib and ≤ 5.1% for N-desmethyl imatinib. The accuracy, expressed as deviation percentage, was found to be within the acceptable range. The recovery data show that the sample preparation method was able to produce consistent, precise, reproducible and absolute recovery for the analytes and IS (>97%). No obvious matrix effects were found for the analytes and IS: the ratios of the peak responses ranged from 85 to 115%, which were within the acceptable limits. The results of accuracy, precision, recovery and matrix effect of imatinib and N

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