Prior to preparation of CRM candidate of chloramphenicol in methanol with a concentration of 100 mg/L, two independent methods including mass balance (MB) and quantitative nuclear magnetic resonance (qNMR) were employed to precisely measure the mass fraction of pure chloramphenicol materials. The mass fraction was assigned to be 99.8% with uncertainty of 0.3%. Homogeneity testing and stability study of chloramphenicol in methanol were examined by using high performance liquid chromatography. Additionally, the uncertainties originating from the process of CRM development were comprehensively evaluated. The experimental results indicate that the property value of this CRM is homogeneous and stable at 4°C for at least six months. The new CRM (GBW(E)082557) can be applicable to calibration of instrument and assurance of accuracy and comparability of results in routine measurement.
A certified reference material (CRM), which is known as one kind of substance or material possessing one or more homogenous and stable property values with uncertainties under a specific confidence level, plays a very important role in the metrological sciences referring to instrument calibration, analytical method validation, and quantity transfer and traceability [
Chloramphenicol, which was a common and famous antibiotic drug, has already been forbidden in the breeding process of livestock and poultry, because the drug residue in agroproducts could lead to toxic side effect to human body through food chain. Therefore, a series of testing standards for determination of chloramphenicol residue in honey, animal products, and animal feed were issued. Meanwhile, chloramphenicol CRMs have been shown to be important and essential in the implementation of these testing standards. Although various commercial CRMs associated with chloramphenicol are available according to the information from COMAR database (international database for certified reference materials), such as purity CRM of chloramphenicol from NIM (National Institute of Metrology, China) and matrix CRMs of chloramphenicol in pork and in honey from ERMM, diversity of chloramphenicol CRMs not only are enrichment for CRM database but also provide more selectable and suitable CRMs for routine work in testing laboratories. Prior to CRM development, a survey of utilization of chloramphenicol CRM in the testing standards was carried out. It was found that the calibration solution was usually prepared individually in each test laboratory by dissolving pure chloramphenicol CRM into methanol or acetonitrile with a concentration of ~100 mg/L. To reduce the operational steps and minimize the error from preparation of calibration solution in each laboratory, a user-friendly and timesaving CRM of chloramphenicol in methanol would be welcomed.
In this paper, the mass fraction of chloramphenicol material was precisely determined by using qNMR approach. To confirm the measurement result, MB as another independent method was carried out on purity determination. As mass fraction of chloramphenicol was accurately assigned, CRM of chloramphenicol in methanol was first developed strictly according to ISO Guide 34 and 35 and JJF 1006-1994 (Chinese Technical Norm of Primary Reference Material) [
All the high purity organic solvents were purchased from Merk. Certified reference materials of ethyl paraben (GBW(E) 100064) with a certificate value of 99.7% as an internal standard for qNMR measurement and chloramphenicol (GBW(E)060907) with a certificate value of 99.8% were supplied by National Institute of Metrology (NIM), China. Methanol-D4 was obtained from Sigma-Aldrich. The powder of chloramphenicol material with a labeled purity of 99.8% was purchased from Tokyo Chemical Industry (TCI) Co., Ltd., Japan.
A high performance liquid chromatography system (Shimadzu, Japan) equipped with Agilent Zorbax XDB-C18 (250 mm × 4.6 mm, 5.0
The purity determination using mass balance method can be described with the following equation:
The main contents and organic impurities of chloramphenicol were determined by using HPLC area normalization method, in which an Agilent Zorbax XDB-C18 (250 × 4.6 mm, 5
The purity of chloramphenicol (
The purity measurement was performed on Bruker Avance III 400 MHz 1H qNMR spectrometer which was equipped with pulsed field gradient probe at temperature of 293.4 K. The setting parameters are as follows: the pulse width of 3.98 s at 90° spectral band width of 8223.43 Hz, delay time of 60 s, and the number of transitions of 16. A sample including 10 mg ethyl paraben CRM as internal standard and 20 mg chloramphenicol was dissolved in 0.75 mL methanol-D4 solvent and was analyzed.
Acquisition on Bruker instrument was based on the command GO. During the NMR acquisition period, a steady-state pulse was applied prior to the relaxation decay. Sixteen dummy scans were acquired before data corrections. The receiver gain was automatically set by instrument. Each sample took about 10 min to finish. Seven samples were measured under the same conditions. All qNMR spectra were analyzed by using MestReNova software. A decay signal was automatically processed by Fourier transformation. The chemical shift of all spectra was referenced to residual 1H signals in deuterated solvent at 3.31 ppm for CD3OD. Phase correction and baseline correction were automatically applied according to the procedure of software guide. Peak integrations were set to an identical range for each spectrum.
According to the determined purity, the CRM of chloramphenicol in methanol was prepared by dissolving precisely weighted 100 mg chloramphenicol into 1000 mL methanol under strictly controlled temperature and moisture. After sufficient mixing and dissolution, the CRM of chloramphenicol in methanol was dispensed into ampoule bottles with 1.0 mL per each. A batch of CRM candidates including about 1000 units was produced and was stored at 4°C condition.
Homogeneity and stability test was carried out by using HPLC-UV approach. 25 bottles were randomly selected from the batch and were examined to test the between-bottle homogeneity of chloramphenicol in methanol. Each bottle was measured in triplicate for testing within-bottle homogeneity. The homogeneity testing results were statistically analyzed by one-way analysis of variance (ANOVA).
The short-term and long-term stability testing was evaluated in eight days and six months, respectively. For short-term stability, the CRM bottles were exposed at 20°C and 40°C on predetermined days of 2, 4, 6, and 8; for long-term stability, the CRM bottles stored at 4°C conditions were analyzed in the 1st, 3rd, and 6th months. A new chloramphenicol calibration solution was freshly prepared when the stability was tested. At each time, a calibration solution and two bottles of CRM were analyzed by HPLC-UV method. The data of stability testing was assessed by regression analysis.
Quantitative nuclear magnetic resonance (qNMR) as a characterization approach for purity measurement was recently recommended by BIPM. To obtain precise and accurate results of purity, several principle rules should be noticed in the measurement processes. Measurement conditions including phase correction, baseline correction, pulse intervals, and spectral window were systematically optimized. First, a certified reference material as an internal standard is necessary to realize the traceability from testing result to SI unit. Second, the selected internal standard and target analyte should be simultaneously soluble in the same and common deuterium labeled solvent. Third, the pointed signal peaks for quantification must have no interference for each other. Ethyl paraben CRM was commonly thought of as an ideal internal standard for purity measurement in many researches; thus, the ethyl paraben was selected in this study. In comparison with spectrum of only ethyl paraben in Figure
Mass fraction of chloramphenicol calculated with different pointed signal peaks (
Pairs | Peak of |
Peak of |
Mean of |
SD/% |
---|---|---|---|---|
A | d | F′ | 99.82 | 0.07 |
B | c | F′ | 99.13 | 0.09 |
C | a | A′ | 98.94 | 0.05 |
“
The H-NMR spectra of (a) internal standard (ethyl paraben and methanol-D4) and (b) sample solution (chloramphenicol, ethyl paraben, and methanol-D4).
Ethyl paraben
Ethyl paraben + chloramphenicol
Baseline enlarged H-NMR spectrum of sample solution (chloramphenicol, ethyl paraben, and methanol-D4).
For further confirmation, mass balance (MB) method was carried out on the measurement of purity. On the contrary with qNMR, MB is an indirect purity measurement method and is accomplished by precise measurements of impurities including organic impurities, moisture, and volatile and nonvolatile impurities. The analysis of organic impurities is generally measured by using chromatography. In this research, HPLC-UV was performed to analyze organic impurities in chloramphenicol. In order to identify and measure impurities sufficiently and comprehensively, methanol (blank) and chloramphenicol in methanol with concentrations of 100 mg/L and 2000 mg/L were determined on the UV absorbance at 280 nm. In comparison with methanol blank, peaks of chloramphenicol and impurities were observed and separated in Figure
Mass fraction of chloramphenicol measured by MB methods.
Sample vials number | HPLC/% | Moisture/% | Purity/% |
---|---|---|---|
1 | 99.87 | 0.020 | 99.85 |
2 | 99.84 | 0.030 | 99.81 |
3 | 99.84 | 0.026 | 99.81 |
4 | 99.87 | 0.025 | 99.85 |
5 | 99.83 | 0.028 | 99.80 |
6 | 99.83 | 0.023 | 99.81 |
7 | 99.82 | 0.024 | 99.80 |
Mean | 99.84 | 0.025 | 99.82 |
SD | 0.020 | 0.003 | 0.02 |
Chromatography of (a) methanol blank, (b) 100 mg/L chloramphenicol in methanol, and (c) 2000 mg/L chloramphenicol in methanol.
For the homogeneity test, 25 ampoule bottles of samples were chosen from the batch of CRMs randomly and were determined in triplicates for each by using optimized HPLC-UV method mentioned in the above section. The area of signal peaks was recorded by HPLC-UV area normalization method as shown in Table
Data of homogeneity study.
Vials number | Peak area | ||||
---|---|---|---|---|---|
1 | 2 | 3 | Mean | SD | |
1 | 3377539 | 3455815 | 3426396 | 3419917 | 39538 |
2 | 3363161 | 3383494 | 3413952 | 3386869 | 25563 |
3 | 3373464 | 3438840 | 3444717 | 3419007 | 39551 |
4 | 3361335 | 3400715 | 3431809 | 3397953 | 35318 |
5 | 3361386 | 3385011 | 3400994 | 3382464 | 19926 |
6 | 3370750 | 3440166 | 3441286 | 3417401 | 40405 |
7 | 3396404 | 3442398 | 3445094 | 3427965 | 27366 |
8 | 3412038 | 3456559 | 3452226 | 3440274 | 24549 |
9 | 3492616 | 3414137 | 3400516 | 3435756 | 49711 |
10 | 3412637 | 3423430 | 3431176 | 3422414 | 9311 |
11 | 3382569 | 3410793 | 3415548 | 3402970 | 17827 |
12 | 3351945 | 3362416 | 3401855 | 3372072 | 26319 |
13 | 3359186 | 3400385 | 3432492 | 3397354 | 36747 |
14 | 3363789 | 3419854 | 3419894 | 3401179 | 32381 |
15 | 3373310 | 3417544 | 3432514 | 3407789 | 30784 |
16 | 3377488 | 3418379 | 3415865 | 3403911 | 22917 |
17 | 3382255 | 3412441 | 3413938 | 3402878 | 17876 |
18 | 3393284 | 3425055 | 3415432 | 3411257 | 16292 |
19 | 3406204 | 3444518 | 3444612 | 3431778 | 22148 |
20 | 3403316 | 3406961 | 3405709 | 3405329 | 1852 |
21 | 3364827 | 3408684 | 3409946 | 3394486 | 25693 |
22 | 3382216 | 3383257 | 3405014 | 3390162 | 12872 |
23 | 3400230 | 3387406 | 3414257 | 3400631 | 13430 |
24 | 3361051 | 3391923 | 3404463 | 3385812 | 22342 |
25 | 3353175 | 3403030 | 3394614 | 3383606 | 26688 |
Results of homogeneity study.
Items | Results |
---|---|
Total mean | 3405649 |
Total SD | 28716 |
Between-bottle | 946997904 |
Within-bottle | 765842476 |
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Evaluation result |
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To assess the effect of temperature change in delivery, portions of samples were prestored at 20°C and 40°C to simulate the shipment conditions. Two ampoule bottles of sample were stored at each temperature for 2, 4, 6, and 8 days. After the indicated storage periods, the samples and a freshly prepared chloramphenicol in methanol as calibration solution were determined in triplicate using the HPLC-UV method. The stability data at 20°C and 40°C were plotted against time, respectively, and the regression lines were calculated (shown in Table
Result of short-term stability study.
Time/day | Value/(mg/L) | |
---|---|---|
Condition/20°C | Condition/40°C | |
0 | 100.7 | 100.7 |
2 | 100.2 | 99.8 |
4 | 100.0 | 96.4 |
6 | 99.4 | 93.3 |
8 | 99.9 | 92.4 |
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Mean | 99.8 | 96.6 |
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−0.119 | −1.153 |
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100.5 | 101.1 |
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0.1053 | 0.7423 |
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0.0513 | 0.1362 |
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3.18 | 3.18 |
Conclusion |
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To satisfy the requirement of CRMs, the chloramphenicol in methanol, which was stored at 4°C, was measured at 1st, 3rd, and 6th months. Long-term stability of chloramphenicol in methanol was examined by using the same analytical method and statistical analysis approach as described in short-term stability study. As shown in Table
Results of long-term stability study.
Time/month | Value/(mg/L) | |||
---|---|---|---|---|
0 | 1 | 3 | 6 | |
#1 | 100.4 | 100.4 | 101.4 | 100.2 |
101.2 | 99.7 | 100.9 | 99.8 | |
101.1 | 99.6 | 101.0 | 100.5 | |
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#2 | 100.2 | 99.1 | 100.5 | 100.3 |
101.6 | 101.4 | 100.3 | 99.9 | |
100.8 | 100.8 | 100.6 | 99.8 | |
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Mean | 100.9 | 100.2 | 100.8 | 100.1 |
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−0.0845 | |||
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100.7 | |||
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0.1801 | |||
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0.0926 | |||
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4.3 | |||
Conclusion |
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In strict accordance with General and Statistical Principles for Characterization of Reference Materials JJF 1343-2012 [
Uncertainty sources in the development process of chloramphenicol in methanol CRM.
For MB method, the standard uncertainty
Additionally, uncertainty of moisture measurement
For qNMR, the standard uncertainty
Therefore, uncertainty of purity measurement was evaluated as follows:
As we mentioned above, chloramphenicol in methanol was prepared by dissolving chloramphenicol into methanol. Thus, the concentration of CRM solution can be calculated by the following equation:
According to above analysis of uncertainty, the evaluated uncertainty results are summarized in Table
Results of uncertainty.
Items | Assessment and calculation | Results |
---|---|---|
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( |
0.41% |
( |
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( |
0.35% |
( |
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( |
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0.28% |
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0.61% |
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0.23% |
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0.56% & 0.41% |
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0.94% |
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2 mg/L |
In order to check up the reliability and accuracy of the assigned value of chloramphenicol in methanol CRM in this development project, an existing certified reference material of chloramphenicol (GBW(E)060907) was selected as calibrator to measure the property value of chloramphenicol in methanol CRM. A calibration solution of chloramphenicol with a concentration of 100.9 mg/L, which is approximate to the assigned value of (
Verification results of chloramphenicol in methanol CRM (unit: mg/L).
Item | Measured value | Assigned value | ||||
---|---|---|---|---|---|---|
1 | 2 | 3 | Mean | SD | ||
CRM | 99.5 | 99.9 | 99.7 | 99.7 | 0.2 | 100 ± 2 |
A user-friendly and time-saving CRM of chloramphenicol in methanol (GBW(E)082557) was successfully developed and was approved by General Administration of Quality Supervision, Inspection, and Quarantine of China. In the study, the qNMR and mass balance methods were employed in the precise measurement of chloramphenicol purity. The homogeneity, stability, and uncertainty of this CRM were sufficiently studied according to the technical specification. In addition, the selection of proton peak in qNMR spectrum for purity measurement was comparatively studied.
The authors declare that there are no competing interests regarding the publication of this paper.
The authors acknowledge the financial support from the Ministry of Agriculture of China (Special Fund for Agro-Scientific Research GJFP201501503) and the Innovation Workshop Project of Chinese Academy of Agricultural Sciences.