An analytical method for separating and detecting ethylbenzene, meta-xylene and p-xylene

Abstract

The present invention relates to an analytical method for separating and detecting ethylbenzene, intermediate xylene and p-xylene, which adopts reverse phase high performance liquid chromatography, uses octadecyl bonded silica gel as a filler in a chromatographic column, uses water as a mobile phase A, uses methanol as a mobile phase B, and performs gradient elution on ethylbenzene. The method can achieve effective separation of ethylbenzene from meta-xylene and p-xylene, and has high detection sensitivity; at the same time, the method of the present invention has the characteristics of accurate and reliable detection results, and provides a reliable reference method for quality research on detecting ethylbenzene, intermediate xylene and p-xylene.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and particularly relates to an analytical method for separating and detecting ethylbenzene, metaxylene and p-xylene. Background Art

[0002] Ethylbenzene: molecular formula is C8H 10 ; The molecular weight is 106.17; The structural formula is as follows:

[0003]

[0004] Meta-xylene: molecular formula is C8H 10 , molecular weight is 106.17, and the structural formula is as follows:

[0005]

[0006] Para-xylene: molecular formula is C8H 10 , molecular weight is 106.17, and the structural formula is as follows:

[0007]

[0008] Meta-xylene and para-xylene are positional isomers of ethylbenzene and have similar chemical properties to ethylbenzene. Under conventional liquid chromatography conditions, their separation is poor and their peaks may even overlap.

[0009] Therefore, it is particularly important to develop an analytical method with high detection sensitivity that can effectively separate the meta-xylene, para-xylene and ethylbenzene peaks. Summary of the invention

[0010] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide an analytical method for separating and detecting ethylbenzene, intermediate xylene and para-xylene, which can achieve effective separation of ethylbenzene from meta-xylene and para-xylene and has high detection sensitivity; at the same time, the method of the present invention has the characteristics of accurate and reliable detection results, and provides a reliable reference method for quality research on detecting ethylbenzene, intermediate xylene and para-xylene.

[0011] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0012] The invention discloses an analytical method for separating and detecting ethylbenzene, metaxylene and p-xylene, which adopts reverse phase high performance liquid chromatography, uses octadecyl bonded silica gel as a filler in a chromatographic column, uses water as a mobile phase A, uses methanol as a mobile phase B, and performs gradient elution on ethylbenzene.

[0013] Preferably, the gradient elution program is as follows:

[0014]

[0015] Preferably, the column temperature of the chromatographic column is 10-50°C, preferably 15°C.

[0016] Preferably, the chromatography column is one of kromasil 100-5-C18.

[0017] Preferably, the inner diameter of the chromatographic column is 3.0-5.0 mm, the length is 100-250 mm, and the filler particle size is 2-5 μm, preferably the inner diameter is 4.6 mm, the length is 250 mm, and the filler particle size is 5 μm.

[0018] Preferably, the injection volume is 1-100 μl, preferably 10 μl.

[0019] Preferably, the flow rate is 1.0 to 1.5 ml / ml, preferably 1.0 ml / ml.

[0020] Preferably, ultraviolet light detection is used with a wavelength of 205 to 220 nm, preferably 210 nm.

[0021] Preferably, the method comprises the following steps:

[0022] (1) Preparation of system test solution: Take ethylbenzene and m-xylene and p-xylene reference solution and dilute with methanol to obtain a system test solution with an ethylbenzene concentration of 1 mg / ml and m-xylene and p-xylene concentrations of 10 μg / ml;

[0023] (2) separation: using reverse phase high performance liquid chromatography, with a chromatographic column using octadecyl bonded silica as a filler, water as a mobile phase A, and methanol as a mobile phase B, to perform gradient elution on the system test solution obtained in step (1);

[0024] (3) Detection: Use ultraviolet light with a wavelength of 205 to 220 nm, preferably 210 nm

[0025] Due to the adoption of the above technical solution, the present invention has the following advantages compared with the prior art:

[0026] 1. The present invention adopts water-methanol reverse phase high performance liquid chromatography to detect ethylbenzene, meta-xylene and p-xylene, and can effectively separate meta-xylene, p-xylene and ethylbenzene peaks; while the HPLC results of other systems and chromatographic columns show that the separation degree of meta-xylene and p-xylene is poor;

[0027] 2. The present invention optimizes and selects the reversed-phase liquid chromatography operating parameters during the detection process, so that m-xylene and p-xylene can be effectively separated while ensuring the specificity, accuracy and sensitivity of the detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the detection spectrum for positioning the intermediate xylene and p-xylene in Example 1;

[0029] Figure 2 This is the detection spectrum for positioning the intermediate xylene and p-xylene in Example 2;

[0030] Figure 3 The detection spectrum of the system test liquid in Example 3;

[0031] Figure 4 The detection spectrum of the system test liquid by the chromatographic column a in Example 4;

[0032] Figure 5 The detection spectrum of the chromatographic column b to the system test liquid in Example 4;

[0033] Figure 6 The detection spectrum of the chromatographic column c to the system test liquid in Example 4;

[0034] Figure 7 The detection spectrum of the system test liquid by the chromatographic column d in Example 4;

[0035] Figure 8 This is the detection spectrum of the system test liquid in Example 5;

[0036] Fig. 9 This is the detection spectrum of the system test liquid in Example 6;

[0037] Fig.10 is the detection spectrum of the system test liquid by gradient a in Example 7;

[0038] Fig.11 This is the detection spectrum of the gradient b on the system test liquid in Example 7;

[0039] Fig.12 is the detection spectrum of the gradient c on the system test liquid in Example 7;

[0040] Fig.13 is the detection spectrum of the gradient d on the system test liquid in Example 7;

[0041] Fig.14 This is the detection spectrum of the system test liquid at a column temperature of 15°C in Example 8;

[0042] Fig.15 This is the detection spectrum of the system test liquid at a column temperature of 20° C. in Example 8;

[0043] Fig.16 This is the detection spectrum of the blank solution in Example 9;

[0044] Fig.17 This is the detection spectrum of the system test liquid in Example 9;

[0045] Fig.18is the detection spectrum of the detection limit solution in Example 9;

[0046] Fig.19 This is the detection spectrum of the quantitative limit solution in Example 9. DETAILED DESCRIPTION

[0047] In order to make the technical solutions and beneficial effects of the present invention more clearly understood, the following is a detailed description by combining the accompanying drawings and listing specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0048] The experimental methods in the following examples without specifying specific conditions are usually based on conventional experimental conditions.

[0049] The equipment and reference substances used in the specific implementation manner of the present invention are all known products, which are obtained by purchasing commercially available products.

[0050] The main instruments used in the specific implementation of the present invention are: high performance liquid chromatograph (model: ThermoU3000, manufacturer: Thermo), electronic balance (model: XSE105DU, manufacturer: Mettler).

[0051] The main samples and reagents used in the specific implementation of the present invention are: ethylbenzene (manufacturer: Aladdin), m-xylene reference solution (concentration: 100 μg / ml, manufacturer: TMRM), p-xylene reference solution (concentration: 100 μg / ml, manufacturer: TMRM), chromatographically pure methanol (TEDIA), and water (Milli-Q).

[0052] Example 1

[0053] Prepare impurity localization solutions: Take m-xylene and p-xylene reference solutions as their respective localization solutions;

[0054] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions were as follows:

[0055] Chromatographic column: Chromatographic column model: Kromasil 100-5C18; Chromatographic column specifications: inner diameter 4.6mm, length 250mm, filler particle size 5μm;

[0056] Mobile phase: water as mobile phase A, methanol as mobile phase B;

[0057] The mobile phase A-mobile phase B (volume ratio of 30:70) was used for isocratic elution for 30 min;

[0058] Detection wavelength: 210nm;

[0059] Column temperature: 25°C;

[0060] Flow rate: 1.0ml / min;

[0061] Injection volume: 10 μl;

[0062] The test results are attached. Figure 1 , meta-xylene and para-xylene are not separated well.

[0063] Example 2

[0064] Prepare impurity localization solutions: Take m-xylene and p-xylene reference solutions as their respective localization solutions;

[0065] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions were as follows:

[0066] Chromatographic column: Chromatographic column model: Welch Ultimate PFP R PFP; chromatographic column specifications: inner diameter 4.6mm, length 250mm, filler particle size 5μm;

[0067] Mobile phase: water as mobile phase A, methanol as mobile phase B;

[0068] Gradient elution was used;

[0069]

[0070]

[0071] Detection wavelength: 210nm;

[0072] Column temperature: 25°C;

[0073] Flow rate: 1.0ml / min;

[0074] Injection volume: 10 μl;

[0075] The test results are attached. Figure 2 , m-xylene and p-xylene overlap.

[0076] Example 3

[0077] Preparation of system test solution: Take 500μl of m-xylene and p-xylene reference solution respectively, place them in a 5ml volumetric flask, add 5mg of ethylbenzene, and dilute to the mark with methanol to obtain a system test solution with an ethylbenzene concentration of 1mg / ml, and m-xylene and p-xylene concentrations of 10μg / ml respectively;

[0078] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions were as follows:

[0079] Chromatographic column: Chromatographic column model: Welch Ultimate PFP R PFP; chromatographic column specifications: inner diameter 4.6mm, length 250mm, filler particle size 5μm;

[0080] Mobile phase: water as mobile phase A, methanol-tetrahydrofuran (volume ratio 80:20) as mobile phase B;

[0081] Gradient elution was used;

[0082]

[0083] Detection wavelength: 210nm;

[0084] Column temperature: 25°C;

[0085] Flow rate: 1.0ml / min;

[0086] Injection volume: 10 μl;

[0087] The test results are attached. Figure 3 , meta-xylene and para-xylene are not separated well.

[0088] Example 4

[0089] Preparation of system test solution: Take 500μl of m-xylene and p-xylene reference solution respectively, place them in a 5ml volumetric flask, add 5mg of ethylbenzene, and dilute to the mark with methanol to obtain a system test solution with an ethylbenzene concentration of 1mg / ml, and m-xylene and p-xylene concentrations of 10μg / ml respectively;

[0090] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions were as follows:

[0091] On the premise of ensuring that other detection conditions are consistent, only the chromatographic column is changed to observe the effects of different chromatographic columns on the separation of m-xylene and p-xylene.

[0092] Chromatographic column a: Chromatographic column model: Kromasil 100-5C18; Chromatographic column specifications: inner diameter 4.6 mm, length 250 mm, filler particle size 5 μm;

[0093] Chromatographic column b: Chromatographic column model: Thermo scientific Acclaim TM 120C18 Specifications of the chromatographic column: inner diameter 4.6 mm, length 250 mm, filler particle size 5 μm;

[0094] Chromatographic column c: Chromatographic column model: Agilent Extend C18; Chromatographic column specifications: inner diameter 4.6 mm, length 250 mm, filler particle size 5 μm;

[0095] Column d: Column model: Welch Ultimate R C18; Column specifications: inner diameter 4.6 mm, length 250 mm, filler particle size 5 μm;

[0096] Mobile phase: water as mobile phase A, methanol-acetonitrile (volume ratio 80:20) as mobile phase B;

[0097] Gradient elution was used;

[0098]

[0099]

[0100] Detection wavelength: 210nm;

[0101] Column temperature: 25°C;

[0102] Flow rate: 1.0ml / min;

[0103] Injection volume: 10 μl;

[0104] The test results are attached. Figure 4-7 The results showed that: using chromatographic column a: the separation degree of m-xylene and p-xylene was 0.51; using chromatographic column b: the separation degree of m-xylene and p-xylene was 0.14; using chromatographic columns c and d, m-xylene and p-xylene overlapped.

[0105] Example 5

[0106] Preparation of system test solution: Take 500μl of m-xylene and p-xylene reference solution respectively, place them in a 5ml volumetric flask, add 5mg of ethylbenzene, and dilute to the mark with methanol to obtain a system test solution with an ethylbenzene concentration of 1mg / ml, and m-xylene and p-xylene concentrations of 10μg / ml respectively;

[0107] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions were as follows:

[0108] Chromatographic column: Chromatographic column model: Kromasil 100-5C18; Chromatographic column specifications: inner diameter 4.6mm, length 250mm, filler particle size 5μm;

[0109] Mobile phase: water as mobile phase A, methanol-acetonitrile (volume ratio 80:20) as mobile phase B;

[0110] Gradient elution was used;

[0111]

[0112] Detection wavelength: 210nm;

[0113] Column temperature: 15°C;

[0114] Flow rate: 1.0ml / min;

[0115] Injection volume: 10 μl;

[0116] The test results are attached. Figure 8 , the separation degree between m-xylene and p-xylene is 0.86.

[0117] Example 6

[0118] Preparation of system test solution: Take 500μl of m-xylene and p-xylene reference solution respectively, place them in a 5ml volumetric flask, add 5mg of ethylbenzene, and dilute to the mark with methanol to obtain a system test solution with an ethylbenzene concentration of 1mg / ml, and m-xylene and p-xylene concentrations of 10μg / ml respectively;

[0119] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions were as follows:

[0120] Chromatographic column: Chromatographic column model: Kromasil 100-5C18; Chromatographic column specifications: inner diameter 4.6mm, length 250mm, filler particle size 5μm;

[0121] Mobile phase: water as mobile phase A, methanol as mobile phase B;

[0122] Gradient elution was used;

[0123]

[0124] Detection wavelength: 210nm;

[0125] Column temperature: 15°C;

[0126] Flow rate: 1.0ml / min;

[0127] Injection volume: 10 μl;

[0128] The test results are attached. Fig. 9 , the separation degree between m-xylene and p-xylene is 0.95.

[0129] Example 7

[0130] Preparation of system test solution: Take 500μl of m-xylene and p-xylene reference solution respectively, place them in a 5ml volumetric flask, add 5mg of ethylbenzene, and dilute to the mark with methanol to obtain a system test solution with an ethylbenzene concentration of 1mg / ml, and m-xylene and p-xylene concentrations of 10μg / ml respectively;

[0131] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions were as follows:

[0132] Under the premise of ensuring that other detection conditions are consistent, only the gradient is changed to observe the impact of the gradient on the entire analytical method.

[0133] Chromatographic column: Chromatographic column model: Kromasil 100-5C18; Chromatographic column specifications: inner diameter 4.6mm, length 250mm, filler particle size 5μm;

[0134] Detection wavelength: 210nm

[0135] Mobile phase: water as mobile phase A, methanol as mobile phase B;

[0136] Gradient a:

[0137]

[0138] Gradient b:

[0139]

[0140] Gradient c:

[0141]

[0142]

[0143] Gradient d:

[0144]

[0145] Column temperature: 15°C

[0146] Flow rate: 1.0ml / min;

[0147] Injection volume: 10 μl;

[0148] The test results are attached. Figure 10-13 The results showed that the separation of m-xylene and p-xylene under the chromatographic conditions of gradient a and gradient b was better than that of gradient c and gradient d. Under the chromatographic conditions of gradient b, the peaks of ethylbenzene, m-xylene and p-xylene appeared earlier, so gradient b was the best choice.

[0149] Example 8

[0150] Preparation of system test solution: Take 500μl of m-xylene and p-xylene reference solution respectively, place them in a 5ml volumetric flask, add 5mg of ethylbenzene, and dilute to the mark with methanol to obtain a system test solution with an ethylbenzene concentration of 1mg / ml, and m-xylene and p-xylene concentrations of 10μg / ml respectively;

[0151] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions were as follows:

[0152] Under the premise of ensuring that other detection conditions are consistent, only the column temperature is changed to observe the impact of the column temperature on the entire analytical method.

[0153] Chromatographic column: Chromatographic column model: Kromasil 100-5C18; Chromatographic column specifications: inner diameter 4.6mm, length 250mm, filler particle size 5μm;

[0154] Detection wavelength: 210nm

[0155] Mobile phase: water as mobile phase A, methanol as mobile phase B;

[0156] Gradient elution was used;

[0157]

[0158] Column temperature: 15°C, 20°C;

[0159] Flow rate: 1.0ml / min;

[0160] Injection volume: 10 μl;

[0161] The test results are attached. Figure 14-15 The results showed that when the column temperature was 15℃, the separation degree of m-xylene and p-xylene was better than that at 20℃, so the column temperature of 15℃ was the best choice.

[0162] Example 9 (Sensitivity Test)

[0163] Blank solution: Take an appropriate amount of diluent to obtain.

[0164] Preparation of system test solution: Take 200μl of m-xylene and p-xylene reference solutions respectively, place them in the same 10ml volumetric flask, add 20mg of ethylbenzene, and dilute to the scale with methanol to obtain a system test solution with an ethylbenzene concentration of 2mg / ml, and m-xylene and p-xylene concentrations of 2μg / ml respectively.

[0165] Preparation of quantitation limit and detection limit solutions: Take appropriate amounts of m-xylene and p-xylene respectively, and prepare the quantitation limit solution at a signal-to-noise ratio of ≥10:1; prepare the detection limit solution at a signal-to-noise ratio of ≥3:1.

[0166] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions were as follows:

[0167] Chromatographic column: Chromatographic column model: Kromasil 100-5C18; Chromatographic column filler: octadecyl bonded silica gel; Chromatographic column specifications: inner diameter 4.6mm, length 250mm, filler particle size 5μm;

[0168] Mobile phase: water as mobile phase A, methanol as mobile phase B;

[0169] Gradient elution was used;

[0170]

[0171] Detection wavelength: 210nm;

[0172] Column temperature: 15°C;

[0173] Flow rate: 1.0ml / min;

[0174] Injection volume: 10 μl;

[0175] The test results are attached. Figure 16-19 The test results are shown in Tables 1 and 2.

[0176] Table 1 Detection limit results

[0177]

[0178] Table 2 Quantitation limit results

[0179]

[0180] From the above results, we can see that the detection limit concentrations of m-xylene and p-xylene are both 0.15μg / ml, and the quantification limit concentrations are both 0.50μg / ml, which shows that the detection sensitivity of this method is good and can meet the detection requirements of ethylbenzene, m-xylene and p-xylene.

[0181] Example 10 (Accuracy Test)

[0182] Preparation of 50% accuracy solution: Take appropriate amount of ethylbenzene, m-xylene and p-xylene reference solution respectively, dilute with methanol to obtain accuracy solution with ethylbenzene concentration of 2 mg / ml and each impurity concentration of 1 μg / ml;

[0183] Preparation of 100% accuracy solution: Take appropriate amount of ethylbenzene and m-xylene and p-xylene reference solution respectively, dilute with methanol to obtain an accuracy solution with ethylbenzene concentration of 2 mg / ml and each impurity concentration of 2 μg / ml;

[0184] Preparation of 150% accuracy solution: Take appropriate amount of ethylbenzene, m-xylene and p-xylene reference solution respectively, dilute with methanol to obtain an accuracy solution with ethylbenzene concentration of 2 mg / ml and each impurity concentration of 3 μg / ml;

[0185] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions were as follows:

[0186] Chromatographic column: Chromatographic column model: Kromasil 100-5C18; Chromatographic column filler: octadecyl bonded silica gel; Chromatographic column specifications: inner diameter 4.6mm, length 250mm, filler particle size 5μm;

[0187] Mobile phase: water as mobile phase A, methanol as mobile phase B;

[0188] Gradient elution was used;

[0189]

[0190] Detection wavelength: 210nm;

[0191] Column temperature: 15°C;

[0192] Flow rate: 1.0ml / min;

[0193] Injection volume: 10 μl;

[0194] The test results are shown in Table 3 below.

[0195] Table 3 Accuracy results

[0196] name Recovery rate range (%) Average recovery rate (%) RSD(%) Meta-Xylene 101.4～105.8 103.9 1.7 p-Xylene 106.3～115.8 109.9 3.2

[0197] From the above results, it can be seen that the recovery rates of various impurities are between 80% and 120%, and the RSD values ​​are all less than 10.0%. The accuracy of this method is good.

[0198] According to the guidelines for validation of analytical methods in Part IV of the 2020 edition of the Chinese Pharmacopoeia, 9101, the sensitivity and accuracy of this method were investigated in detail. The validation results met the predetermined acceptable standards, ensuring the specificity, accuracy and sensitivity of the detection method, proving that this method is suitable for the separation and detection of ethylbenzene, metaxylene and paraxylene.

[0199] In summary, the present invention adopts water as mobile phase A, methanol as mobile phase B, and performs gradient elution on ethylbenzene. The separation degree between meta-xylene and para-xylene is good. The detection result of the method of the present invention is accurate and reliable, and a method is provided for controlling the quality research of ethylbenzene, meta-xylene and para-xylene.

[0200] It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. An analytical method for separating and detecting ethylbenzene, meta-xylene and para-xylene, characterized in that: Reverse phase high performance liquid chromatography was used, with water as mobile phase A, methanol as mobile phase B, and ethylbenzene as gradient elution; Wherein, the gradient elution procedure is as follows: ； The chromatographic column was kromasil 100-5-C18; Use UV light for detection.

2. The analytical method for separating and detecting ethylbenzene, meta-xylene and para-xylene according to claim 1, characterized in that: The column temperature of the chromatographic column is 10-50°C.

3. The analytical method for separating and detecting ethylbenzene, intermediate xylene and paraxylene according to claim 2, characterized in that: The column temperature of the chromatographic column is 15°C.

4. The analytical method for separating and detecting ethylbenzene, meta-xylene and para-xylene according to claim 1, characterized in that: The inner diameter of the chromatographic column is 3.0-5.0 mm, the length is 100-250 mm, and the filler particle size is 2-5 μm.

5. The analytical method for separating and detecting ethylbenzene, meta-xylene and para-xylene according to claim 4, characterized in that: The inner diameter of the chromatographic column is 4.6 mm, the length is 250 mm, and the filler particle size is 5 μm.

6. The analytical method for separating and detecting ethylbenzene, meta-xylene and para-xylene according to claim 1, characterized in that: The injection volume of the reversed-phase high performance liquid chromatography is 1-100 μl.

7. The analytical method for separating and detecting ethylbenzene, meta-xylene and para-xylene according to claim 6, characterized in that: The injection volume of the reverse phase high performance liquid chromatography was 10 μl.

8. The analytical method for separating and detecting ethylbenzene, meta-xylene and para-xylene according to claim 1, characterized in that: The flow rate of the mobile phase is 1.0-1.5 ml / min.

9. The analytical method for separating and detecting ethylbenzene, meta-xylene and p-xylene according to claim 8, characterized in that: The flow rate of the mobile phase is 1.0 ml / min.

10. The analytical method for separating and detecting ethylbenzene, meta-xylene and para-xylene according to claim 1, characterized in that: Use ultraviolet light with a wavelength of 205~220nm for detection.

11. The analytical method for separating and detecting ethylbenzene, meta-xylene and para-xylene according to claim 10, characterized in that: Use ultraviolet light with a wavelength of 210 nm for detection.

12. The analytical method for separating and detecting ethylbenzene, meta-xylene and p-xylene according to any one of claims 1 to 11, characterized in that: The steps include: (1) Preparation of system test solution: Take ethylbenzene and m-xylene and p-xylene reference solution and dilute them with methanol to obtain a system test solution with an ethylbenzene concentration of 1 mg / ml and m-xylene and p-xylene concentrations of 10 μg / ml; (2) Separation: using reverse phase high performance liquid chromatography, with water as mobile phase A and methanol as mobile phase B, to perform gradient elution on the system test solution obtained in step (1); (3) Detection.

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