Detection method of isopropyl acetoacetate related substances

Abstract

The invention relates to the technical field of chemical analysis, and particularly discloses a method for detecting isopropyl acetoacetate related substances. According to the method, Astec CHIRRADEX B-DM (0.25 mm * 30m * 0.12 [mu] m) is adopted as a detection chromatographic column, the column temperature is set to be 38-42 DEG C and maintained for 4-6 min, then the temperature is increased to 175-185 DEG C at the rate of 4-6 DEG C / min and maintained for 1-3 min, the temperature of a sample inlet is 115-125 DEG C, the temperature of a detector is 245-255 DEG C, and the split ratio is (5-10): 1, so that simultaneous quantitative detection of multiple impurities in isopropyl acetoacetate is realized. The method provided by the invention is more beneficial to truly reflecting the quality of isopropyl acetoacetate, is beneficial to monitoring the synthesis process of isopropyl acetoacetate, is further beneficial to improving the quality of dipine drugs and reducing the drug use risk, and has a relatively high practical value.

Description

Technical Field

[0001] The invention relates to the technical field of chemical analysis, and in particular to a method for detecting isopropyl acetoacetate-related substances. Background Art

[0002] Isopropyl acetoacetate (IAA) is a colorless transparent liquid and an important organic chemical raw material. It is widely used in the fields of synthetic drugs, fragrances, dyes and pesticides, and is mainly used in the synthesis of dihydropyridine drugs.

[0003] At present, the industry generally adopts the diketene method to prepare isopropyl acetoacetate, that is, diketene and isopropanol are catalyzed by acid, and the reflux reaction prepares isopropyl acetoacetate, and the synthesis process route is as follows. The isopropyl acetoacetate synthesized by this route inevitably contains various impurities, such as related substances such as isopropyl acetate, diketene, acetic anhydride, methyl acetoacetate and ethyl acetoacetate. As the starting material of dihydrochloric acid drugs, the quality of isopropyl acetoacetate directly affects the purity of downstream products. Therefore, the presence of the above-mentioned various impurities will directly affect the subsequent reaction, thereby causing the product purity to be low, and increasing the safety risk of dihydrochloric acid drugs. Therefore, a detection method for related substances in isopropyl acetoacetate is developed to fully detect the impurity situation in isopropyl acetoacetate, which is of great significance to effectively control the purity and quality safety of dihydrochloric acid drugs.

[0004] Summary of the invention

[0005] Aiming at the technical problem that there is a lack of effective detection of related substances in isopropyl acetoacetate in the prior art, the present invention provides a method for detecting related substances in isopropyl acetoacetate.

[0006] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0007] A method for detecting related substances of isopropyl acetoacetate, the detection method being a gas chromatography method, comprising the following steps:

[0008] (1) Preparation of test solution and mixed reference solution:

[0009] Preparation of mixed reference solution: Take isopropyl acetate, diketene, acetic anhydride, methyl acetoacetate and ethyl acetoacetate reference substances and prepare a mixed reference solution with a solvent;

[0010] Preparation of test solution: Take a sample of isopropyl acetoacetate and prepare a test solution with solvent;

[0011] (2) Take the mixed reference solution and test solution and perform the test under the following gas chromatography conditions:

[0012] Detector: Hydrogen flame ionization detector;

[0013] Chromatographic column: Astec CHIRALDEX B-DM, 0.25 mm × 30 m × 0.12 μm;

[0014] Column temperature: 38℃-42℃ for 4min-6min, then increase the temperature to 175℃-185℃ at a rate of 4℃ / min-6℃ / min, and maintain for 1min-3min;

[0015] Inlet temperature: 115℃-125℃;

[0016] Detector temperature: 245℃-255℃;

[0017] Split ratio: 5-15:1.

[0018] The isopropyl acetoacetate-related substances described in the present invention refer to isopropyl acetate, diketene, acetic anhydride, methyl acetoacetate and ethyl acetoacetate.

[0019] Compared with the prior art, the detection method of isopropyl acetoacetate-related substances provided by the invention adopts gas chromatography, takes Astec CHIRALDEX B-DM (0.25 mm×30 m×0.12 μm) as a detection chromatographic column, cooperates with a specific programmed temperature rising mode and specific gas chromatographic conditions such as injection port temperature, detector temperature and split ratio, realizes simultaneous quantitative detection of multiple known impurities and unknown impurities in isopropyl acetoacetate, is more conducive to truly reflecting the quality of isopropyl acetoacetate, can be used as a qualitative and quantitative detection method for multiple impurities in the synthesis process of isopropyl acetoacetate, and provides a technical basis for improving the production process and determining product quality.

[0020] Furthermore, the detection method of isopropyl acetoacetate-related substances provided by the present invention has been verified by methodology to be highly specific, accurate, sensitive, repeatable and stable, and easy to operate. The method can achieve qualitative and quantitative analysis of various impurities in isopropyl acetoacetate through one detection, thus filling the gap that the prior art cannot effectively detect the quality of isopropyl acetoacetate, and is beneficial to achieving quality control of dihydropyridine drug products (such as the cardiovascular drug nimodipine), thereby helping to reduce the drug safety risk of dihydropyridine drugs.

[0021] Preferably, the column temperature is: maintained at 40°C for 5 min, then raised to 180°C at a rate of 5°C / min, and maintained for 2 min.

[0022] The preferred programmed temperature rising method combined with a specific chromatographic column can make the peak shapes of isopropyl acetoacetate and various impurities good, and is conducive to improving the separation between isopropyl acetoacetate and various impurities, thereby realizing accurate quantitative analysis of various impurities in isopropyl acetoacetate.

[0023] Preferably, the injection port temperature is 120°C.

[0024] The optimal combination of column temperature and injection port temperature is conducive to ensuring effective separation of samples, as well as ensuring the peak shapes of the main components and various impurities, and improving the accuracy and sensitivity of detection.

[0025] Preferably, the detector temperature is 250°C.

[0026] The preferred detector temperature can ensure the stability of the baseline and reduce the baseline noise, thereby improving the accuracy and sensitivity of the detection results.

[0027] Preferably, the split ratio is 10:1.

[0028] Preferably, the carrier gas is nitrogen with a flow rate of 0.8 mL / min-1.2 mL / min.

[0029] More preferably, the carrier gas flow rate is 1.0 mL / min.

[0030] Preferably, the injection volume is 1.0 μL.

[0031] Preferably, the solvent is acetonitrile.

[0032] Preferably, the concentration of isopropyl acetate in the mixed reference solution is 125 μg / mL, the concentration of diketene is 125 μg / mL, the concentration of acetic anhydride is 125 μg / mL, the concentration of methyl acetoacetate is 125 μg / mL, and the concentration of ethyl acetoacetate is 25 μg / mL.

[0033] Preferably, the concentration of the test solution is 25 mg / mL.

[0034] The preferred gas chromatography conditions are conducive to fully detecting known impurities and unknown impurities in isopropyl acetoacetate, and realizing quantitative detection of multiple impurities in isopropyl acetoacetate.

[0035] The gas chromatography detection method provided by the present invention can realize the effective detection of various impurities in isopropyl acetoacetate, and through methodological research and verification, it is proved that the method of the present invention has good sensitivity, accuracy and reproducibility, fills the gap that the prior art cannot effectively detect the quality of isopropyl acetoacetate, is also conducive to the monitoring of the synthesis process of isopropyl acetoacetate, and is further conducive to improving the quality of dihydropyridine drugs, reducing the risk of drug use, and has high practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The gas chromatogram of the blank solvent (acetonitrile) under the specificity item in Example 2 of the present invention;

[0037] Figure 2 The gas chromatogram of the mixed reference solution under the specific property item in Example 2 of the present invention; wherein, peak 1 is isopropyl acetate, peak 2 is diketene, peak 3 is acetic anhydride, peak 4 is methyl acetoacetate, and peak 5 is ethyl acetoacetate;

[0038] Figure 3 The gas chromatogram of the test solution under the specificity item in Example 2 of the present invention;

[0039] Figure 4 The gas chromatogram of the spiked test solution under the specificity item in Example 2 of the present invention, wherein peak 1 is isopropyl acetate, peak 2 is diketene, peak 3 is acetic anhydride, peak 4 is methyl acetoacetate, peak 5 is ethyl acetoacetate, and peak 6 is isopropyl acetoacetate;

[0040] Figure 5 It is a gas chromatogram of the spiked test solution in Comparative Example 1 of the present invention, wherein peak 1 is isopropyl acetate, peak 2 is diketene, peak 3 is acetic anhydride, peak 4 is methyl acetoacetate, peak 5 is ethyl acetoacetate, and peak 6 is isopropyl acetoacetate;

[0041] Figure 6 The gas chromatogram of the spiked test solution and the mixed reference stock solution in Comparative Example 2 of the present invention, wherein peak 1 is isopropyl acetate, peak 2 is diketene, peak 3 is acetic anhydride, peak 4 is methyl acetoacetate, peak 5 is ethyl acetoacetate, and peak 6 is isopropyl acetoacetate;

[0042] Figure 7 The gas chromatogram of the spiked test solution in Comparative Example 3 of the present invention. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0044] Example 1

[0045] 1.1 Solution preparation:

[0046] Blank solvent: acetonitrile.

[0047] Preparation of mixed reference stock solution: Take appropriate amounts of isopropyl acetate, diketene, acetic anhydride, methyl acetoacetate and ethyl acetoacetate reference substances, accurately weigh them, and use acetonitrile to make a mixed solution containing approximately 1250μg, 1250μg, 1250μg, 1250μg and 250μg of each in 1mL as the mixed reference stock solution.

[0048] Mixed reference solution: Take the mixed reference stock solution prepared above and quantitatively dilute it with acetonitrile to prepare a mixed solution containing approximately 125 μg of isopropyl acetate, 125 μg of diketene, 125 μg of acetic anhydride, 125 μg of methyl acetoacetate, and 25 μg of ethyl acetoacetate per 1 mL as the mixed reference solution.

[0049] Preparation of test solution: Take about 250 mg of isopropyl acetoacetate sample, accurately weigh it, place it in a 10 mL volumetric flask, and use acetonitrile to prepare a test solution with a concentration of 25 mg / mL.

[0050] Preparation of spiked test solution: Take about 250 mg of isopropyl acetoacetate sample, accurately weigh it, place it in a 10 mL volumetric flask, add 1 mL of the mixed reference stock solution prepared above, add acetonitrile to dilute to the scale, shake well, and obtain the spiked test solution.

[0051] 1.2 Gas chromatography conditions:

[0052] Detector: Hydrogen flame ionization (FID) detector;

[0053] Chromatographic column: Astec CHIRALDEX B-DM, 0.25 mm × 30 m × 0.12 μm;

[0054] Column temperature: 40°C for 5 min, then raised to 180°C at a rate of 5°C / min and maintained for 2 min;

[0055] Inlet temperature: 120°C;

[0056] Detector temperature: 250°C;

[0057] Carrier gas: high-purity nitrogen (purity 99.99%), flow rate 1 mL / min;

[0058] Split ratio: 10:1;

[0059] Injection volume: 1.0 μL.

[0060] Example 2

[0061] Methodological validation:

[0062] 2.1 Exclusivity

[0063] Take blank solvent, the mixed reference solution, the test solution and the spiked test solution and test them according to the above gas chromatography conditions, record the chromatogram, and the results are as follows: Figure 1-Figure 4 As shown, Figure 1 is the chromatogram of blank solvent, Figure 2 is the chromatogram of the mixed reference solution. Figure 3 is the chromatogram of the test solution, Figure 4 The chromatogram of the spiked test solution is shown in Table 1.

[0064] Table 1 Separation of components in spiked test solution

[0065]

[0066]

[0067] It can be seen from the above table that the blank solvent in the detection method provided by this scheme does not interfere with the detection of each component, and the separation between the components meets the requirements, indicating that this method has good specificity.

[0068] 2.2 Limit of detection and limit of quantification

[0069] Take appropriate amounts of isopropyl acetoacetate, isopropyl acetate, diketene, acetic anhydride, methyl acetoacetate and ethyl acetoacetate reference substances, weigh them accurately, and use acetonitrile to prepare mixed solutions containing about 100 μg of each in 1 mL. Accurately measure the above mixed solutions, perform serial dilutions, inject them into the gas chromatograph, and record the chromatograms. When the chromatographic peak height is about 3 times the baseline noise, it is the detection limit; when the chromatographic peak height is about 10 times the baseline noise, it is the quantitative limit. The results are shown in Table 2. And prepare 6 portions of reference substance solutions according to the quantitative limit concentration, test them according to the above gas chromatography conditions, and record the peak area of ​​each chromatographic peak. The results are shown in Table 3.

[0070] Table 2 Detection limit and quantification limit test results

[0071]

[0072] Table 3 Results of repeatability test of limit of quantitation

[0073]

[0074]

[0075] The test results show that the maximum RSD of the peak area of ​​each component measured 6 times is 9.90%, indicating that this method has high sensitivity and good repeatability of the quantitative limit.

[0076] 2.3 Linear range

[0077] Accurately weigh the isopropyl acetoacetate reference substance and each impurity reference substance, and prepare a series of linear solutions with acetonitrile. Accurately measure 1.0 μL of each, inject and test according to the above gas chromatography conditions, record the chromatogram, determine the peak area, and draw a linear regression curve with concentration (μg / mL) as the horizontal axis and peak area as the vertical axis. The results are shown in Tables 4 to 9.

[0078] Table 4 Isopropyl acetate linear test results

[0079]

[0080] Table 5 Diketene linear test results

[0081]

[0082] Table 6 Acetic anhydride linear test results

[0083]

[0084]

[0085] Table 7 Linearity test results of methyl acetoacetate

[0086]

[0087] Table 8 Ethyl acetoacetate linear test results

[0088]

[0089] Table 9 Isopropyl acetoacetate linear test results

[0090]

[0091] The above results show that the linear relationship between the components is good, and r is greater than 0.9982, indicating that the method provided by this scheme has a good linear relationship.

[0092] 2.4 Recovery rate

[0093] Take appropriate amount of isopropyl acetate, diketene, acetic anhydride, methyl acetoacetate and ethyl acetoacetate reference substances, accurately weigh them, and quantitatively dilute them with acetonitrile to prepare a mixed stock solution containing approximately 1250μg of isopropyl acetate, 1250μg of diketene, 1250μg of acetic anhydride, 1250μg of methyl acetoacetate, and 500μg of ethyl acetoacetate per 1mL; take about 250mg of isopropyl acetoacetate sample, accurately weigh it, place it in a 10mL volumetric flask, prepare 9 portions in parallel, then accurately add appropriate volume of the mixed stock solution to each of them, make up to the scale with acetonitrile, and prepare 3 portions in parallel for each concentration as low, medium and high recovery test solutions.

[0094] The above 9 recovery solutions were taken and tested respectively according to the above gas chromatography conditions, the chromatograms were recorded, and the recovery rates were calculated. The results are shown in Tables 10 to 14.

[0095] Table 10 isopropyl acetate recovery test results

[0096]

[0097] Table 11 Diketene recovery test results

[0098]

[0099] Table 12 Acetic anhydride recovery test results

[0100]

[0101] Table 13 methyl acetoacetate recovery test results

[0102]

[0103] Table 14 Ethyl acetoacetate recovery test results

[0104]

[0105]

[0106] The test results show that the recovery rate of each component is between 80% and 120%, and the maximum RSD is 7.53%, indicating that the method provided by the present invention has good accuracy.

[0107] 2.5 Repeatability

[0108] Take 6 samples of isopropyl acetoacetate and prepare them into test solutions with a concentration of 25 mg / mL with acetonitrile. The solutions were tested according to the above-mentioned gas chromatography conditions. The results are shown in Table 15.

[0109] Table 15 Repeatability test results

[0110]

[0111] The test results showed that the results of the 6 test solutions were basically consistent, indicating that this method has good repeatability.

[0112] 2.6 Solution stability

[0113] Accurately weigh appropriate amounts of isopropyl acetoacetate and various impurity reference substances, and prepare test solution and mixed reference substance solution according to the above method. Sampling was performed at 0 h, 2 h, 4 h, 8 h, and 14 h, respectively, and the results were tested according to the above-mentioned gas chromatography conditions. The results are shown in Tables 16 and 17.

[0114] Table 16 Stability results of mixed reference solution

[0115] Element 0h 2h 4h 8h 12h RSD% Isopropyl acetate 32857 32390 32207 35920 32678 4.62 Diketene 19069 17960 18915 18854 18937 2.38 Acetic anhydride 13240 13492 13245 13078 13778 2.05 Methyl acetoacetate 26884 25979 26231 26820 26518 1.45 Ethyl acetoacetate 6437 6120 6704 6269 7134 6.12

[0116] Table 17 Stability results of test solution

[0117] Element 0h 2h 4h 8h 14h Isopropyl acetate Not detected Not detected Not detected Not detected Not detected Diketene Not detected Not detected Not detected Not detected Not detected Acetic anhydride Not detected Not detected Not detected Not detected Not detected Methyl acetoacetate Not detected Not detected Not detected Not detected Not detected Ethyl acetoacetate 0.011 0.016 0.014 0.011 0.017 purity% 99.964 99.958 99.964 99.966 99.959 Other individual impurities% 0.017 0.016 0.016 0.016 0.017

[0118] The test results show that after being placed at room temperature for 14 hours, the RSD of the peak area of ​​each component is less than 10%; there is no significant difference in the detection results of the test solution at each time point.

[0119] 2.7 Durability

[0120] Accurately weigh each impurity reference substance and prepare a mixed reference solution as described above. Change the initial temperature, heating rate, injection port temperature, and detector temperature of the column temperature respectively. Perform detection according to the gas chromatography detection conditions of the present invention. The results are shown in Tables 18 to 21.

[0121] Table 18 Separation degree at different initial temperatures

[0122] condition Initial temperature 40℃ Initial temperature 38℃ Initial temperature 42℃ Acetonitrile -- -- -- Isopropyl acetate 6.693 7.069 6.994 Diketene 5.574 5.748 6.720 Acetic anhydride 12.448 13.83 11.103 Methyl acetoacetate 10.883 10.378 11.107 Ethyl acetoacetate 4.401 4.044 4.395

[0123] Table 19 Separation degree at different heating rates

[0124] condition Heating rate 5℃ / min Heating rate: 4.5℃ / min Heating rate: 5.5℃ / min Acetonitrile -- -- -- Isopropyl acetate 6.693 7.799 7.740 Diketene 5.574 7.891 7.788 Acetic anhydride 12.448 14.926 14.337 Methyl acetoacetate 10.883 11.365 10.987 Ethyl acetoacetate 4.401 4.439 4.264

[0125] Table 20 Separation of different injection port temperatures

[0126] condition Inlet temperature 120℃ Inlet temperature 115°C Inlet temperature 125℃ Acetonitrile -- -- -- Isopropyl acetate 6.693 6.453 7.343 Diketene 5.574 5.571 6.754 Acetic anhydride 12.448 12.426 12.466 Methyl acetoacetate 10.883 10.59 10.760 Ethyl acetoacetate 4.401 4.212 4.374

[0127] Table 21 Separation of different detector temperatures

[0128] condition Detector 250℃ Detector 245℃ Detector 255℃ Acetonitrile -- -- -- Isopropyl acetate 6.693 7.705 6.752 Diketene 5.574 6.833 5.516 Acetic anhydride 12.448 12.718 12.925 Methyl acetoacetate 10.883 11.319 10.735 Ethyl acetoacetate 4.401 4.443 4.128

[0129] It can be seen from the above table that by changing the conditions, the separation degree of each component meets the requirements, indicating that the method provided by this scheme has good durability.

[0130] Comparative Example 1

[0131] This comparative example provides a method for detecting substances related to isopropyl acetoacetate, and the detection method is exactly the same as the gas chromatography conditions under item 1.2 in Example 1, the only difference being that the chromatographic column in the chromatographic conditions is a capillary column ZB-1701 (30m×0.53mm×1.0μm) with 14% cyanopropylphenyl-86% dimethylpolysiloxane (or similar polarity) as the stationary liquid, and other conditions remain unchanged.

[0132] Take the spiked test solution under Example 1 and inject it for testing, record the chromatogram, and the result is as follows Figure 5shown.

[0133] The test results show that the main component peak of isopropyl acetoacetate is extended and does not achieve baseline separation with ethyl acetoacetate, resulting in low detection accuracy of ethyl acetoacetate.

[0134] Comparative Example 2

[0135] This comparative example provides a method for detecting substances related to isopropyl acetoacetate, and the detection method is exactly the same as the gas chromatography conditions under item 1.2 in Example 1, the only difference being that the chromatographic column in the chromatographic conditions is a capillary column HP-INNOWAX (60m×0.32mm×0.5μm) with cross-linked polyethylene glycol (or similar polarity) as the stationary liquid, and other conditions remain unchanged.

[0136] Take the spiked test solution and the mixed reference stock solution under Example 1 for testing, record the chromatogram, and the results are as follows: Figure 6 shown.

[0137] The test results show that ethyl acetoacetate completely overlaps with the main component isopropyl acetoacetate.

[0138] Comparative Example 3

[0139] This comparative example provides a method for detecting substances related to isopropyl acetoacetate, and the detection method is exactly the same as the gas chromatography conditions under item 1.2 in Example 1, the only difference being that the chromatographic column in the chromatographic conditions is a capillary column HP-50+ (30m×0.53mm×1μm) with 50% phenyl-50% dimethylpolysiloxane (or similar polarity) as the stationary liquid, and other conditions remain unchanged.

[0140] Take the spiked test solution under Example 1 and inject it for testing, record the chromatogram, and the result is as follows Figure 7 shown.

[0141] The test results show that the main component, isopropyl acetoacetate, has a double peak (retention time is 6.033min and 7.883min), and the related substances cannot be detected.

[0142] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting related substances of isopropyl acetoacetate, characterized in that: The detection method is gas chromatography, comprising the following steps: (1) Preparation of test solution and mixed reference solution: Preparation of mixed reference solution: Take isopropyl acetate, diketene, acetic anhydride, methyl acetoacetate and ethyl acetoacetate reference substances and prepare a mixed reference solution with a solvent; Preparation of test solution: Take a sample of isopropyl acetoacetate and prepare a test solution with solvent; (2) Take the mixed reference solution and test solution and perform the test under the following gas chromatography conditions: Detector: Hydrogen flame ionization detector; Chromatographic column: Astec CHIRALDEX B-DM, 0.25 mm × 30 m × 0.12 μm; Column temperature: 38℃-42℃ for 4min-6min, then increase the temperature to 175℃-185℃ at a rate of 4℃ / min-6℃ / min, and maintain for 1min-3min; Inlet temperature: 115℃-125℃; Detector temperature: 245℃-255℃; Split ratio: 5-15:

1.

2. The method for detecting related substances of isopropyl acetoacetate as claimed in claim 1, characterized in that: The column temperature was maintained at 40°C for 5 min, then raised to 180°C at a rate of 5°C / min and maintained for 2 min.

3. The method for detecting related substances of isopropyl acetoacetate as claimed in claim 1, characterized in that: The injection port temperature is 120°C.

4. The method for detecting related substances of isopropyl acetoacetate as claimed in claim 1, characterized in that: Detector temperature 250°C.

5. The method for detecting related substances of isopropyl acetoacetate as claimed in claim 1, characterized in that: The split ratio is 10:

1.

6. The method for detecting related substances of isopropyl acetoacetate as claimed in claim 1, characterized in that: The carrier gas was nitrogen with a flow rate of 0.8 mL / min-1.2 mL / min.

7. The method for detecting related substances of isopropyl acetoacetate according to claim 6, wherein: The carrier gas flow rate was 1.0 mL / min.

8. The method for detecting related substances of isopropyl acetoacetate as claimed in claim 1, characterized in that: The injection volume was 1.0 μL.

9. The method for detecting related substances of isopropyl acetoacetate according to claim 1, wherein: The concentration of isopropyl acetate in the mixed reference solution is 125 μg / mL, the concentration of diketene is 125 μg / mL, the concentration of acetic anhydride is 125 μg / mL, the concentration of methyl acetoacetate is 125 μg / mL, and the concentration of ethyl acetoacetate is 25 μg / mL.

10. The method for detecting related substances of isopropyl acetoacetate according to claim 1, characterized in that: The concentration of the test solution is 25 mg / mL.

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