Method for detecting nitrosamine impurities in metoclopramide hydrochloride

Abstract

The invention provides a method for detecting nitrosamine impurities in metoclopramide hydrochloride. A liquid chromatography-mass spectrometry method is adopted. The liquid chromatographic conditions are as follows: a Waters Xbridege BEH C18 chromatographic column is adopted, and octadecylsilane chemically bonded silica is adopted as a filling agent; the column temperature is 40 DEG C; a 0.1% formic acid aqueous solution is used as a mobile phase A, methanol is used as a mobile phase B, and gradient elution is carried out; the flow rate is 0.6 ml / min; the temperature of a sample feeding disc is 5 DEG C; the sample injection volume is 10 microliters; mass spectrum conditions are as follows: an ESI source is adopted; a positive ion mode and an MRM are adopted as a scanning mode; the positive ion voltage is 4000 V; the sheath gas is 40Arb; the auxiliary gas is 20Arb; the purging gas is 0Arb; the temperature of an ion transmission tube is 300 DEG C; the temperature of the vaporizer is 300 DEG C; according to the method, the content of nitrosamine impurities in metoclopramide hydrochloride can be quantitatively detected at high sensitivity, so that the quality of metoclopramide hydrochloride is controlled, and the medication safety is ensured.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis and detection, and particularly to a method for detecting nitrosamine impurities in metoclopramide hydrochloride. Background Art

[0002] Metoclopramide hydrochloride is an antiemetic drug that can be used for chemotherapy, radiotherapy, surgery, craniocerebral injury, sequelae of brain trauma, naval operations, and vomiting caused by drugs; it can also be used for symptomatic treatment of nausea and vomiting symptoms in various diseases such as acute gastroenteritis, biliary pancreas, and uremia; it can also be used before diagnostic duodenal intubation to help with smooth intubation.

[0003] Currently, in the process of synthesizing metoclopramide hydrochloride, there may be residues of secondary amine impurities. The residual secondary amine impurities are likely to react with sodium nitrite (NaNO2) or other nitrosating reagents, thereby generating nitrosamine impurities. Nitrosamine impurities belong to the "substances of concern" mentioned in the ICH M7(R1) "Evaluating and Controlling DNA-Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk" guideline and have carcinogenic risks. For chemical drugs, the risks of nitrosamine impurities in the drugs should be fully evaluated, and the levels of nitrosamine impurities should be controlled below the safety limit.

[0004] Therefore, a highly sensitive method is needed to detect "nitrosamine impurities" in metoclopramide hydrochloride. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for detecting nitrosamine impurities in metoclopramide hydrochloride, which can achieve highly sensitive quantitative detection of the content of nitrosamine impurities in metoclopramide hydrochloride, control the quality of metoclopramide hydrochloride drugs, and ensure the safety of drug use.

[0006] The present invention adopts the following technical solutions to solve the above technical problems:

[0007] A method for detecting nitrosamine impurities in metoclopramide hydrochloride, using liquid chromatography-tandem mass spectrometry (LC-MS / MS);

[0008] Among them, the liquid chromatography conditions are as follows: using a Waters Xbridege BEH C18 column with a size of 150mm×4.6mm and a particle size of 3μm, with octadecylsilyl-bonded silica gel as the filler; the column temperature is 40°C; 0.1% formic acid aqueous solution is used as mobile phase A, and methanol is used as mobile phase B for gradient elution; the flow rate is 0.6ml / min; the injection tray temperature is 5°C; the injection volume is 10μl;

[0009] The mass spectrometry conditions are as follows: The ion source uses the ESI source; the scanning mode uses the positive ion mode, multiple reaction monitoring (MRM); the positive ion voltage is 4000 V; the sheath gas is 40 Arb; the auxiliary gas is 20 Arb; the purge gas is 0 Arb; the ion transfer tube temperature is 300 °C; the vaporizer temperature is 300 °C.

[0010] As one of the preferred embodiments of the present invention, in the gradient elution of the liquid chromatography, the proportions of the mobile phases are as follows:

[0011] At 0 min, mobile phase A is 70% and mobile phase B is 30%;

[0012] At 3.0 min, mobile phase A is 70% and mobile phase B is 30%;

[0013] At 6.5 min, mobile phase A is 45% and mobile phase B is 55%;

[0014] At 6.6 min, mobile phase A is 10% and mobile phase B is 90%;

[0015] At 12.0 min, mobile phase A is 10% and mobile phase B is 90%;

[0016] At 12.1 min, mobile phase A is 70% and mobile phase B is 30%;

[0017] At 17.0 min, mobile phase A is 70% and mobile phase B is 30%.

[0018] As one of the preferred embodiments of the present invention, the acquisition time of the mass spectrometry is: 2 min to 3 min, 8 min to 12 min.

[0019] As one of the preferred embodiments of the present invention, it includes the following specific steps:

[0020] (1) Solution preparation

[0021] Test solution: Take the raw material of metoclopramide hydrochloride and dilute it with a diluent to obtain the test solution;

[0022] Reference solution: Take the raw material of the impurity and dilute it with a diluent to obtain the reference solution;

[0023] (2) Liquid chromatography - mass spectrometry analysis

[0024] Take the reference solution and the test solution and inject them into the liquid chromatography - mass spectrometry combined instrument respectively;

[0025] The liquid chromatography conditions were as follows: A Waters Xbridege BEH C18 column with dimensions of 150 mm × 4.6 mm and a particle size of 3 μm, packed with octadecylsilyl silica gel, was used; the column temperature was 40°C; an aqueous solution of 0.1% formic acid was used as mobile phase A, and methanol was used as mobile phase B for gradient elution; the flow rate was 0.6 ml / min; the injection tray temperature was 5°C; the injection volume was 10 μl.

[0026] The mass spectrometry conditions were as follows: The electrospray ionization (ESI) source was used; the positive ion mode and multiple reaction monitoring (MRM) were adopted; the positive ion voltage was 4000 V; the sheath gas was 40 Arb (argon); the auxiliary gas was 20 Arb; the purge gas was 0 Arb; the temperature of the ion transfer tube was 300°C; the temperature of the vaporizer was 300°C.

[0027] The chromatogram was recorded, and the contents of each component were calculated.

[0028] As one of the preferred embodiments of the present invention, in step (1), the diluent was prepared by mixing methanol, water, and formic acid in a volume ratio of "500:500:1".

[0029] As one of the preferred embodiments of the present invention, in step (1), the concentration of the test solution was 1 mg / ml.

[0030] As one of the preferred embodiments of the present invention, in the reference solution of step (1), the impurity raw materials included methyl 2-methoxy-4-(methylnitrosamino)benzoate, 1-nitrosopiperazine, and N-nitroso desethyl metoclopramide.

[0031] As one of the preferred embodiments of the present invention, in the reference solution, the final concentration of methyl 2-methoxy-4-(methylnitrosamino)benzoate was 3.3 ng / ml, 1-nitrosopiperazine was 13.3 ng / ml, and N-nitroso desethyl metoclopramide was 0.6 ng / ml.

[0032] As one of the preferred embodiments of the present invention, in step (2), the contents of each component were calculated by the external standard method based on the peak area.

[0033] The advantages of the present invention compared with the prior art are as follows:

[0034] The present invention uses liquid chromatography - mass spectrometry (LC-MS) and, by selecting appropriate liquid chromatography and mass spectrometry conditions, can achieve rapid, accurate, and highly sensitive detection of nitrosamine impurities such as methyl 2-methoxy-4-(methylnitrosamino)benzoate, 1-nitrosopiperazine, and N-nitroso desethyl metoclopramide in metoclopramide hydrochloride, thus controlling the quality of metoclopramide hydrochloride drugs and ensuring medication safety. Description of the Drawings

[0035] Figure 1It is the linear relationship diagram of methyl 2-methoxy-4-(methylnitrosamino)benzoate in Experimental Example 1;

[0036] Figure 2 It is the linear relationship diagram of 1-nitrosopiperazine in Experimental Example 1;

[0037] Figure 3 It is the linear relationship diagram of N-nitroso-desethyl metoclopramide in Experimental Example 1. Detailed implementation manners

[0038] The embodiments of the present invention will be described in detail below. These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments. The raw materials, reagents, equipment and methods used in the following embodiments, unless otherwise specified, are all conventional raw materials, reagents, equipment and methods in the art, and will not be elaborated.

[0039] Meanwhile, the structural information of metoclopramide hydrochloride and each nitrosamine impurity of the present invention is shown in Table 1.

[0040] Table 1. Structural information of metoclopramide hydrochloride and each nitrosamine impurity

[0041]

[0042]

[0043] Example 1

[0044] A method for detecting nitrosamine impurities in metoclopramide hydrochloride in this example includes the following steps:

[0045] (1) Solution preparation

[0046] ① Diluent (blank solution): Mix methanol, water and formic acid in a volume ratio of 500:500:1.

[0047] ② Test solution: Accurately weigh 10 mg of metoclopramide hydrochloride raw material, place it in a 10-ml volumetric flask, dissolve it with the diluent and dilute it to the 10-ml scale, shake well, and obtain it (the concentration of metoclopramide hydrochloride is 1 mg / ml).

[0048] ③ Reference solution:

[0049] Accurately weigh 2 mg of methyl 2-methoxy-4(methylnitrosamino)benzoate reference substance, place it in a 20-ml volumetric flask, dissolve it with methanol and dilute it to the 20-ml scale, shake well, and use it as reference stock solution A;

[0050] Accurately weigh 2 mg of 1-nitrosopiperazine reference substance, place it in a 20-ml volumetric flask, dissolve it with methanol and dilute to the 20-ml mark, shake well to obtain reference stock solution B;

[0051] Accurately weigh 2 mg of N-nitroso-desethyl metoclopramide reference substance, accurately weigh it, place it in a 20-ml volumetric flask, dissolve it with methanol and dilute to the 20-ml mark, shake well to obtain reference stock solution C:

[0052] Take 33 μl of the above reference stock solution A, 133 μl of reference stock solution B and 6 μl of reference stock solution C, place them in the same 10-ml volumetric flask, dilute to the 10-ml mark with diluent, shake well to obtain reference stock solution D;

[0053] Take 100 μl of reference stock solution D, place it in a 10-ml volumetric flask, dilute to the 10-ml mark with diluent, shake well to obtain the final required reference solution.

[0054] In the final reference solution, the concentration of methyl 2-methoxy-4-(methylnitrosamino)benzoate is 3.3 ng / ml, the concentration of 1-nitrosopiperazine is 13.3 ng / ml, and the concentration of N-nitroso-desethyl metoclopramide is 0.6 ng / ml.

[0055] (2) Liquid chromatography-mass spectrometry analysis

[0056] Take 10 μl each of the reference solution and the test solution, inject them into a liquid chromatography-mass spectrometry instrument respectively, and record the chromatogram. If there are target impurity peaks in the chromatogram of the test solution, calculate according to the external standard method with the peak area

[0057] Among them, the liquid chromatography conditions are as follows:

[0058] Use a Waters Xbridege BEH C18 column (150 mm × 4.6 mm, 3 μm), with octadecylsilyl-bonded silica gel as the packing material; the column temperature is 40 °C; use 0.1% formic acid aqueous solution as mobile phase A and methanol as mobile phase B; the flow rate is 0.6 ml / min; the injection tray temperature is 5 °C; the injection volume is 10 μl; elute according to the program in Table 2.

[0059] Table 2. Elution program (mobile phase ratio, volume percentage)

[0060]

[0061]

[0062] The mass spectrometry conditions are as follows:

[0063] The ion source is an ESI source; the scanning mode is the positive ion mode (Positive), multiple reaction monitoring MRM; the positive ion voltage (Positive Ion) is 4000 V; the sheath gas (Sheath Gas) is 40 Arb; the auxiliary gas (Aux Gas) is 20 Arb; the sweep gas (SwoopGas) is 0 Arb; the temperature of the ion transfer tube (Ions Transfer tube temp) is 300 °C; the temperature of the vaporizer (VaporizerTemp) is 300 °C; operate according to the mass spectrometry parameters in Table 3.

[0064] Table 3. Mass Spectrometry Parameters

[0065]

[0066] The mass spectrometry acquisition time is: 2 min to 3 min, 8 min to 12 min.

[0067] Experimental Example 1

[0068] This experimental example is used to verify the feasibility and application advantages of the method of the present invention.

[0069] The instrument and chromatographic conditions are the same as those in Example 1.

[0070] I. System Suitability

[0071] The diluent (blank solution) and the reference substance solution are prepared in the same manner as in Example 1.

[0072] The reference substance solution is injected continuously for 6 times, and the RSD of the peak areas and the recovery rates of each impurity peak are shown in Tables � to 6.

[0073] Table 4. System Suitability Results of Methyl 2-Methoxy-4-(Methylnitrosoamino)Benzoate

[0074]

[0075] Table 5. System Suitability Results of 1-Nitrosopiperazine

[0076]

[0077] Table 6. System Suitability Results of N-Nitroso-Desethyl-Metoclopramide

[0078]

[0079]

[0080] From the above results, it can be seen that the blank solution has no interference on the detection of the target nitrosamine impurities; for the reference solution, six consecutive injections were made, and the RSD of the peak area of methyl 2-methoxy-4-(methylnitrosamino)benzoate was 2.2%, the RSD of the peak area of 1-nitrosopiperazine was 0.5%, and the RSD of the peak area of N-nitroso-desethyl metoclopramide was 4.2%. The RSD of each peak area was less than 5%, indicating that the system suitability of this method is good.

[0081] II. Specificity

[0082] The diluent (blank solution), reference solution, and test solution were prepared in the same manner as in Example 1.

[0083] Spiked test solution: Take 10 mg of metoclopramide hydrochloride raw material and weigh it accurately, place it in a 10-ml volumetric flask; at the same time, accurately measure 100 μl of the reference stock solution D and place it in the same volumetric flask; add diluent to dissolve and dilute to the 10-ml mark, shake well, and you will get it.

[0084] According to the detection conditions in Example 1, inject the blank solution, reference solution, test solution, and spiked test solution respectively, record the chromatogram, and the specificity results are shown in Table 7.

[0085] Table 7. Specificity Results

[0086]

[0087] The above results show that there are no other interfering peaks in the sample that affect the detection of the target impurities, and the test sample does not affect the retention time of the target impurity peak, indicating that the specificity of this method is good.

[0088] III. Detection Limit and Quantitation Limit

[0089] The diluent (blank solution) and reference solution were prepared in the same manner as in Example 1.

[0090] Detection limit solution: Accurately measure 5 μl of the reference stock solution D and place it in a 10-ml volumetric flask, add diluent to dilute to the 10-ml mark, shake well, and you will get it.

[0091] Quantitation limit solution: Accurately measure 10 μl of the reference stock solution D and place it in a 10-ml volumetric flask, add diluent to dilute to the 10-ml mark, shake well, and you will get it.

[0092] According to the detection method in Example 1, inject the detection limit solution and quantitation limit solution respectively, and record the chromatogram. The detection limit and quantitation limit results of each target impurity are shown in Table 8 and Table 9.

[0093] Table 8. Detection Limit Results

[0094]

[0095] Table 9. Quantitation Limit Results

[0096]

[0097]

[0098]

[0099] The above results show that the detection limits and quantitative limit concentrations of each target impurity meet the limit requirements, with high detection sensitivity. The RSD of the peak areas of each target impurity at the quantitative limit concentration is less than 5%, meeting the requirements.

[0100] IV. Linear Relationship Test

[0101] The diluent (blank solution) and the reference solution were prepared in the same manner as in Example 1.

[0102] Preparation of the linear solution: Accurately pipette the reference stock solution D according to Table 10, place it in a 10-ml volumetric flask, dilute it to the 10-ml mark with the diluent, and shake well to obtain the linear solution.

[0103] Table 10. Preparation Table of the Linear Solution

[0104]

[0105] Accurately pipette 10 μl of each of the above linear solutions with different concentrations and inject them into the liquid chromatograph. Record the chromatogram, and plot a standard curve with the peak area as the ordinate and the concentration as the abscissa. The results are shown in Tables 11 to 13 and Figures 1 to 3 .

[0106] Table 11. Results of the Linear Relationship of Methyl 2-Methoxy-4-(Methylnitrosamino)benzoate

[0107] Equivalent limit level 10％ 50％ 100％ 150％ 200％ Concentration (ng / ml) 0.3278 1.6391 3.2782 4.9173 6.5563 Peak area (A) 431 2781 4287 6383 8696

[0108] Table 12. Results of the Linear Relationship of 1-Nitrosopiperazine

[0109] Equivalent limit level 10％ 50％ 100％ 150％ 200％ Concentration (ng / ml) 1.3219 6.6093 13.2186 19.8278 26.4371 Peak area (A) 583 3031 6293 9297 12698

[0110] Table 13. Results of the Linear Relationship Test of N-Nitroso-Desethyl Metoclopramide

[0111] Equivalent limit level 10％ 50％ 100％ 150％ 200％ Concentration (ng / ml) 0.0599 0.2996 0.5991 0.8987 1.1983 Peak area (A) 104 495 949 1415 1914

[0112] The above results show that each nitrosamine impurity shows a good linear relationship within the range of the quantitative limit to 200% of the limit, and the linear correlation coefficient r is greater than 0.999, meeting the requirements.

[0113] V. Accuracy

[0114] The diluent (blank solution), the reference solution, and the test solution were prepared in the same manner as in Example 1.

[0115] LOQ spiked test solution: Accurately weigh 10 mg of metoclopramide hydrochloride raw material, place it in a 10-ml volumetric flask. At the same time, accurately measure 10 μl of reference stock solution D and place it in the same volumetric flask; dissolve with diluent and dilute to the 10-ml mark, shake well to obtain. Prepare 3 parallel portions.

[0116] 100% spiked test solution: Accurately weigh 10 mg of metoclopramide hydrochloride raw material, place it in a 10-ml volumetric flask. At the same time, accurately measure 100 μl of reference stock solution D and place it in the same volumetric flask; dissolve with diluent and dilute to the 10-ml mark, shake well to obtain. Prepare 3 parallel portions.

[0117] 150% spiked test solution: Accurately weigh 10 mg of metoclopramide hydrochloride raw material, place it in a 10-ml volumetric flask. At the same time, accurately measure 150 μl of reference stock solution D and place it in the same volumetric flask; dissolve with diluent and dilute to the 10-ml mark, shake well to obtain. Prepare 3 parallel portions.

[0118] According to the chromatographic conditions and detection methods in Example 1, take 10 μl of each of the above solutions, calculate the content of each target impurity in each test sample by the external standard method based on the peak area, and calculate the recovery rate. The results are shown in Tables 14 - 16.

[0119] Table 14, Recovery rate of methyl 2-methoxy-4-(methylnitrosamino)benzoate

[0120]

[0121] Table 15, Recovery rate of 1-nitrosopiperazine

[0122]

[0123] Table 16, Recovery rate of N-nitroso-desethylmetoclopramide

[0124]

[0125] From the above results, it can be seen that: the recovery rates of methyl 2-methoxy-4-(methylnitrosamino)benzoate are all between 94.5% and 102.7%, the average recovery rate is 99.4%, the RSD is 2.64%, and the recovery rate is good; the recovery rates of 1-nitrosopiperazine are all between 90.7% and 98.3%, the average recovery rate is 94.76%, the RSD is 2.36%, and the recovery rate is good; the recovery rates of N-nitroso-desethylmetoclopramide are all between 95.1% and 105.8%, the average recovery rate is 99.79%, the RSD is 4.01%, and the recovery rate is good.

[0126] In summary, the detection method provided by the present invention has strong specificity and high sensitivity, and can accurately detect nitrosamine impurities such as methyl 2-methoxy-4-(methylnitrosamino)benzoate, 1-nitrosopiperidine, and N-nitroso-desethyl metoclopramide in metoclopramide hydrochloride. The detection results are accurate and reliable, and can more scientifically control the quality of metoclopramide hydrochloride.

[0127] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting nitrosamine impurities in metoclopramide hydrochloride, characterized in that, Liquid chromatography-mass spectrometry (LC-MS) was used; Among them, the liquid chromatography conditions were as follows: A Waters Xbridege BEH C18 column with dimensions of 150 mm × 4.6 mm and 3 μm, packed with octadecylsilyl-bonded silica gel, was used; the column temperature was 40 °C; an aqueous solution of 0.1% formic acid was used as mobile phase A, and methanol was used as mobile phase B for gradient elution; the flow rate was 0.6 ml / min; the injection tray temperature was 5 °C; the injection volume was 10 μl; The mass spectrometry conditions were as follows: The ion source used was the ESI source; the scan mode was the positive ion mode, and multiple reaction monitoring (MRM) was used; the positive ion voltage was 4000 V; the sheath gas was 40 Arb; the auxiliary gas was 20 Arb; the purge gas was 0 Arb; the ion transfer tube temperature was 300 °C; the vaporizer temperature was 300 °C.

2. The method for detecting nitrosamine impurities in metoclopramide hydrochloride according to claim 1, characterized in that, In the gradient elution of the liquid chromatography, the mobile phase ratios were as follows: At 0 min, mobile phase A was 70% and mobile phase B was 30%; At 3.0 min, mobile phase A was 70% and mobile phase B was 30%; At 6.5 min, mobile phase A was 45% and mobile phase B was 55%; At 6.6 min, mobile phase A was 10% and mobile phase B was 90%; At 12.0 min, mobile phase A was 10% and mobile phase B was 90%; At 12.1 min, mobile phase A was 70% and mobile phase B was 30%; At 17.0 min, mobile phase A was 70% and mobile phase B was 30%.

3. The method for detecting nitrosamine impurities in metoclopramide hydrochloride according to claim 1, wherein The acquisition time of the mass spectrometry was: 2 min to 3 min, 8 min to 12 min.

4. The method for detecting nitrosamine impurities in metoclopramide hydrochloride according to any one of claims 1 to 3, characterized in that, It included the following specific steps: (1) Solution preparation Test solution: Take the metoclopramide hydrochloride raw material drug and dilute it with a diluent to obtain the test solution; Reference solution: Take the impurity raw materials and dilute them with a diluent to obtain the reference solution; (2) Liquid chromatography-mass spectrometry analysis Take the reference solution and the test solution and inject them into the liquid chromatography-mass spectrometer respectively; The liquid chromatography conditions were as follows: A Waters Xbridege BEH C18 column with dimensions of 150 mm × 4.6 mm and 3 μm, packed with octadecylsilyl-bonded silica gel, was used; the column temperature was 40 °C; an aqueous solution of 0.1% formic acid was used as mobile phase A, and methanol was used as mobile phase B for gradient elution; the flow rate was 0.6 ml / min; the injection tray temperature was 5 °C; the injection volume was 10 μl; The mass spectrometry conditions were as follows: The ion source used was the ESI source; the scan mode was the positive ion mode, and multiple reaction monitoring (MRM) was used; the positive ion voltage was 4000 V; The sheath gas was 40 Arb; the auxiliary gas was 20 Arb; The purge gas was 0 Arb; the ion transfer tube temperature was 300 °C; the vaporizer temperature was 300 °C; Record the chromatogram and calculate the content of each component.

5. The method for detecting nitrosamine impurities in metoclopramide hydrochloride according to claim 4, wherein In the step (1), the preparation method of the diluent was: Mix methanol, water and formic acid in a volume ratio of "500:500:1" to obtain it.

6. The method for detecting nitrosamine impurities in metoclopramide hydrochloride according to claim 4, characterized in that, In the step (1), the concentration of the test solution was 1 mg / ml.

7. The method for detecting nitrosamine impurities in metoclopramide hydrochloride according to claim 4, characterized in that, In the reference solution of the step (1), the impurity raw materials included methyl dimethoxy-4-(methylnitrosoamino)benzoate, 1-nitrosopiperazine and N-nitroso-desethyl metoclopramide.

8. The method for detecting nitrosamine impurities in metoclopramide hydrochloride according to claim 7, wherein In the reference substance solution, the final concentration of methyl 2-methoxy-4-(methylnitrosamino)benzoate is 3.3 ng / ml, that of 1-nitrosopiperazine is 13.3 ng / ml, and that of N-nitroso desethyl metoclopramide is 0.6 ng / ml.

9. The method for detecting nitrosamine impurities in metoclopramide hydrochloride according to claim 4, wherein In the step (2), the contents of each component are calculated by the external standard method based on the peak area.

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