Method for detecting dissolution of degradation impurities of levodopa and carbidopa in carlevodopa sustained release tablet

Abstract

The invention relates to the technical field of pharmaceutical analysis, and discloses a dissolution detection method for degradation impurities of levodopa and carbidopa in a carbidopa-levodopa sustained release tablet, which comprises the following steps: step 1, detecting by adopting liquid chromatography, and using a specific chromatographic column to achieve high separation efficiency according to gradient elution; 2, a mobile phase is composed of ethanol and a trifluoroacetic acid solution according to a specific ratio; and step 3, taking a proper amount of carlevodopa sustained-release tablets, carrying out a dissolution curve experiment, respectively taking a dissolution solution at each time point, filtering, discarding 1ml, taking a subsequent filtrate, and diluting the subsequent filtrate with a 0.1 mol / L hydrochloric acid solution (50: 50). The invention relates to a dissolution detection method for degradation impurities of levodopa and carbidopa in a carlevodopa sustained release tablet, which can be used for dissolution detection of levodopa, carbidopa and degradation impurities thereof, and has the advantages of strong specificity, high sensitivity, good precision and the like. The quality of the raw material medicines of levodopa and carbidopa and the carbidopa-levodidopa sustained release tablet is ensured, and the safety of clinical medication is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis, and specifically to a dissolution detection method for levodopa and carbidopa degradation impurities in carbidopa-levodopa sustained-release tablets. Background Art

[0002] Parkinson's disease, like Alzheimer's disease, is a common neurodegenerative disease that impairs patients' motor, language, and other functional abilities. The current cause is still unclear. Research has found that patients lack dopamine in the basal ganglia. Currently, the main treatments are drugs that control dopamine secretion or brain stimulation with electric current. Dopamine cannot directly enter the brain. Levodopa, as the direct metabolic precursor of dopamine, can cross the blood-brain barrier and enter the central nervous system, increasing the dopamine in the brain and reducing the patient's movement disorders. However, levodopa is rapidly converted into dopamine outside the brain, which leads to waste of levodopa and an increase in side effects in patients. Carbidopa has a strong inhibitory effect on dopa decarboxylase. When administered in combination with levodopa, it can inhibit the conversion of levodopa by dopa decarboxylase, increasing the amount of levodopa entering the brain, reducing the clinical dosage of levodopa (standard maintenance dose 1.5 - 3.0 g / day), and reducing the gastrointestinal reactions frequently caused by the large amount of levodopa. Currently, carbidopa-levodopa sustained-release tablets have such efficacy.

[0003] Currently, there are few reports on the dissolution detection method for carbidopa-levodopa sustained-release tablets. For finished preparations, there are situations of separation of two main components, interference between two main components, and interference from excipients, and carbidopa will degrade during the dissolution experiment, making it more difficult to detect levodopa, carbidopa, and their degradation impurities.

[0004] Based on this, the present invention provides a dissolution detection method for levodopa and carbidopa degradation impurities in carbidopa-levodopa sustained-release tablets. Summary of the Invention

[0005] The present invention provides a dissolution detection method for levodopa and carbidopa degradation impurities in carbidopa-levodopa sustained-release tablets, which can effectively quantitatively detect levodopa, carbidopa, and their degradation impurities in carbidopa-levodopa sustained-release tablets, and effectively improve the accuracy of detection by investigating degradation impurities and determining the impurity correction factors in different media. This detection method can objectively, simply, and accurately evaluate the dissolution of levodopa and carbidopa in carbidopa-levodopa sustained-release tablets, ensuring the safety of clinical medication.

[0006] The present invention provides the following technical solution: A dissolution detection method for levodopa and carbidopa degradation impurities in carbidopa-levodopa sustained-release tablets, comprising the following steps:

[0007] Step 1: Use liquid chromatography for detection. Using a specific chromatographic column for gradient elution has high separation efficiency;

[0008] Step 2: The mobile phase consists of ethanol and trifluoroacetic acid solution in a specific ratio;

[0009] Step 3: Take an appropriate amount of Carbidopa and Levodopa Sustained Release Tablets, conduct a dissolution curve experiment, take the dissolution solutions at each time point, filter, discard 1 ml, and take the subsequent filtrate: dilute with 0.1 mol / L hydrochloric acid solution (50:50) as the test solution;

[0010] Step 4: Take appropriate amounts of levodopa and carbidopa reference substances, accurately weigh, dissolve and dilute with 0.1 mol / L hydrochloric acid to prepare a solution containing about 0.03 mg of carbidopa and about 0.11 mg of levodopa per 1 ml as the reference solution;

[0011] Step 5: Obtain the chromatograms of the test solution and the reference solution under qualified chromatographic separation conditions, and determine the dissolution of levodopa and carbidopa by the external standard method.

[0012] Preferably, the chromatographic column for the liquid chromatography detection is packed with octadecylsilane chemically bonded silica gel.

[0013] Preferably, the mobile phase for the liquid chromatography detection includes mobile phase A and mobile phase B. Mobile phase A is 0.1% trifluoroacetic acid solution; mobile phase B is ethanol. Among them, the concentration of the trifluoroacetic acid solution can be 0.005%, 0.05%, 0.2%, 0.5%, etc., but is not limited to the values listed above. Other unlisted values within the above value range are equally applicable.

[0014] Preferably, the flow rate of the mobile phase for the liquid chromatography detection is 0.8 - 1.2 ml / min, and the column temperature is 25 - 45 °C. Among them, the flow rate of the mobile phase can be 0.8 ml / min, 0.9 ml / min, 1.0 ml / min, 1.1 ml / min, 1.2 ml / min, etc., and the column temperature can be 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, etc., but is not limited to the values listed above. Other unlisted values within the above value range are equally applicable.

[0015] Preferably, the detection wavelength for the liquid chromatography detection is 260 - 300 nm.

[0016] Preferably, the elution program for the liquid chromatography detection is isocratic elution, mobile phase A: mobile phase B - 90:10, but is not limited to the values listed above. Other unlisted values within the above value range are equally applicable, which can effectively retain the chromatograms of levodopa, carbidopa and their degradation impurity peaks, and effectively improve the detection sensitivity.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] Dissolution detection method for levodopa and carbidopa degradation impurities in the controlled release tablet of combined levodopa and carbidopa. The detection method in this solution can perform dissolution detection on levodopa, carbidopa and their degradation impurities, and has the advantages of strong specificity, high sensitivity, good precision, etc., ensuring the quality of levodopa, carbidopa bulk drugs and the controlled release tablet of combined levodopa and carbidopa, and improving the safety of clinical medication. Description of the Drawings

[0019] Figure 1 For the reference preparation in Example 1 of the present invention, it was placed in 0.1 mol / L hydrochloric acid solution at 37 °C for 20 hours;

[0020] Figure 2 For the reference preparation in Example 1 of the present invention, it was placed in pH 4.5 acetate buffer solution at 37 °C for 20 hours;

[0021] Figure 3 For the reference preparation in Example 1 of the present invention, it was placed in pH 6.8 phosphate buffer solution at 37 °C for 20 hours;

[0022] Figure 4 For the reference preparation in Example 1 of the present invention, it was placed in aqueous solution at 37 °C for 20 hours;

[0023] Figure 5 Chromatogram at the limit of quantitation of carbidopa in pH 4.5 acetate buffer solution in Example 2 of the present invention;

[0024] Figure 6 Chromatogram at 160% concentration level of carbidopa in pH 4.5 acetate buffer solution in Example 2 of the present invention;

[0025] Figure 7 Chromatogram at the limit of quantitation of impurity G of carbidopa in pH 4.5 acetate buffer solution in Example 2 of the present invention;

[0026] Figure 8 Chromatogram at 200% concentration level of impurity G of carbidopa in pH 4.5 acetate buffer solution in Example 2 of the present invention;

[0027] Figure 9 Chromatogram at the limit of quantitation of α-methyl-3,4-dihydroxyphenylpropionic acid in pH 4.5 acetate buffer solution in Example 2 of the present invention;

[0028] Figure 10 Chromatogram at 200% concentration level of α-methyl-3,4-dihydroxyphenylpropionic acid in pH 4.5 acetate buffer solution in Example 2 of the present invention;

[0029] Figure 11It is the chromatogram of the limit of quantitation of carbidopa in pH 6.8 phosphate buffer in Example 3 of the present invention;

[0030] Figure 12 It is the chromatogram of carbidopa at 160% concentration level in pH 6.8 phosphate buffer in Example 3 of the present invention;

[0031] Figure 13 It is the chromatogram of the limit of quantitation of impurity G of carbidopa in pH 6.8 phosphate buffer in Example 3 of the present invention;

[0032] Figure 14 It is the chromatogram of impurity G of carbidopa at 200% concentration level in pH 6.8 phosphate buffer in Example 3 of the present invention;

[0033] Figure 15 It is the chromatogram of the limit of quantitation of α-methyl-3,4-dihydroxyphenylpropionic acid in pH 6.8 phosphate buffer in Example 3 of the present invention;

[0034] Figure 16 It is the chromatogram of α-methyl-3,4-dihydroxyphenylpropionic acid at 200% concentration level in pH 6.8 phosphate buffer in Example 3 of the present invention;

[0035] Figure 17 It is the chromatogram of the limit of quantitation of carbidopa in aqueous solution in Example 4 of the present invention;

[0036] Figure 18 It is the chromatogram of carbidopa at 160% concentration level in aqueous solution in Example 4 of the present invention;

[0037] Figure 19 It is the chromatogram of the limit of quantitation of impurity G of carbidopa in aqueous solution in Example 4 of the present invention;

[0038] Figure 20 It is the chromatogram of impurity G of carbidopa at 200% concentration level in aqueous solution in Example 4 of the present invention;

[0039] Figure 21 It is the chromatogram of the limit of quantitation of α-methyl-3,4-dihydroxyphenylpropionic acid in aqueous solution in Example 4 of the present invention;

[0040] Figure 22 It is the chromatogram of α-methyl-3,4-dihydroxyphenylpropionic acid at 200% concentration level in aqueous solution in Example 4 of the present invention;

[0041] Figure 23 It is the linear relationship diagram of carbidopa in pH 4.5 acetate buffer in Example 2 of the present invention;

[0042] Figure 24It is the linear relationship diagram of carbidopa impurity G in pH 4.5 acetate buffer solution in Example 2 of the present invention;

[0043] Figure 25 It is the linear relationship diagram of α-methyl-3,4-dihydroxyphenylpropionic acid in pH 4.5 acetate buffer solution in Example 2 of the present invention;

[0044] Figure 26 It is the linear relationship diagram of carbidopa in pH 6.8 phosphate buffer solution in Example 3 of the present invention;

[0045] Figure 27 It is the linear relationship diagram of carbidopa impurity G in pH 6.8 phosphate buffer solution in Example 3 of the present invention;

[0046] Figure 28 It is the linear relationship diagram of α-methyl-3,4-dihydroxyphenylpropionic acid in pH 6.8 phosphate buffer solution in Example 3 of the present invention;

[0047] Figure 29 It is the linear relationship diagram of carbidopa in aqueous medium in Example 4 of the present invention;

[0048] Figure 30 It is the linear relationship diagram of carbidopa impurity G in aqueous medium in Example 4 of the present invention;

[0049] Figure 31 It is the linear relationship diagram of α-methyl-3,4-dihydroxyphenylpropionic acid in aqueous medium in Example 4 of the present invention. Detailed implementation manners

[0050] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0051] Please refer to Figure 1-22 , the dissolution detection method for levodopa and carbidopa degradation impurities in carbidopa-levodopa sustained-release tablets, includes the following steps:

[0052] Step 1: Use liquid chromatography for detection, and use a specific chromatographic column for gradient elution with high separation efficiency;

[0053] Step 2: The mobile phase is composed of ethanol and trifluoroacetic acid solution in a specific ratio;

[0054] Step 3: Take an appropriate amount of Carbidopa and Levodopa Sustained Release Tablets, conduct a dissolution curve experiment, take the dissolution solutions at each time point, filter, discard 1 ml, and take the subsequent filtrate: dilute with 0.1 mol / L hydrochloric acid solution (50:50) as the test solution;

[0055] Step 4: Take appropriate amounts of levodopa and carbidopa reference substances, accurately weigh them, dissolve and dilute with 0.1 mol / L hydrochloric acid to prepare a solution containing about 0.03 mg of carbidopa and about 0.11 mg of levodopa per 1 ml as the reference solution;

[0056] Step 5: Obtain the chromatograms of the test solution and the reference solution under qualified chromatographic separation conditions, and determine the dissolution of levodopa and carbidopa by the external standard method.

[0057] Among them; the chromatographic column for liquid chromatography detection is packed with octadecylsilane chemically bonded silica gel.

[0058] Among them; the mobile phase for liquid chromatography detection includes mobile phase A and mobile phase B. Mobile phase A is 0.1% trifluoroacetic acid solution; mobile phase B is ethanol. Among them, the concentration of the trifluoroacetic acid solution can be 0.005%, 0.05%, 0.2% or 0.5%, etc., but is not limited to the values listed above. Other unlisted values within the above value range are equally applicable.

[0059] Among them; the flow rate of the mobile phase for liquid chromatography detection is 0.8 - 1.2 ml / min, and the column temperature is 25 - 45 °C. Among them, the flow rate of the mobile phase can be 0.8 ml / min, 0.9 ml / min, 1.0 ml / min, 1.1 ml / min or 1.2 ml / min, etc., and the column temperature can be 25 °C, 30 °C, 35 °C, 40 °C or 45 °C, etc., but is not limited to the values listed above. Other unlisted values within the above value range are equally applicable.

[0060] Among them; the detection wavelength for liquid chromatography detection is 260 - 300 nm.

[0061] Among them; the elution program for liquid chromatography detection is isocratic elution, mobile phase A: mobile phase B - 90:10, but is not limited to the values listed above. Other unlisted values within the above value range are equally applicable.

[0062] Example 1

[0063] A detection method for determining the dissolution of levodopa and carbidopa in levodopa and carbidopa raw materials or preparations by HPLC, comprising the following steps:

[0064] Step 1: Prepare the test solution: Crush one tablet of the reference preparation and place it in 900 ml of medium (0.1 mol / L hydrochloric acid solution, pH 4.5 acetate buffer solution, pH 6.8 phosphate buffer solution, and water). Sonicate for 30 minutes, stir with a glass rod during sonication, sample and filter it into a liquid phase vial as the 0-hour sample. Then place it in a dissolution apparatus at 37°C, sample at 3 hours and 20 hours respectively, filter to obtain the 3-hour and 20-hour samples. Take the solutions at each time point, filter, discard 1 ml, and take the subsequent filtrate: Dilute it with 0.1 mol / L hydrochloric acid solution (50:50) as the test solution;

[0065] Step 2: Obtain the chromatogram of the test solution under qualified chromatographic separation conditions, determine the degradation impurities through the chromatogram, and determine the mass conservation of levodopa and carbidopa through the total peak area;

[0066] Chromatographic conditions:

[0067] Chromatographic column: Use octadecylsilane-bonded silica gel as the packing material for chromatographic conditions (Waters Atlantis T3 4.6×250 mm, 5 μm). Use sodium dihydrogen phosphate (take 6 g of anhydrous sodium dihydrogen phosphate, dissolve it in water and dilute to 1000 ml, adjust the pH value to 2.2 with phosphoric acid) and anhydrous ethanol (95:5) as phase A, and anhydrous ethanol as phase B for gradient elution. The column temperature is at room temperature, the sample tray temperature is 6°C, the flow rate is 1.0 ml per minute, and the detection wavelength is 280 nm; The injection volume is 20 μl;

[0068] Elution gradient

[0069] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 40 100 0 70 70 30 80 100 0 90 100 0

[0070] The results of the dissolution degradation test are shown in the following table:

[0071] Table 1 Results of the dissolution degradation test

[0072]

[0073]

[0074] Conclusion: It can be seen from the chromatogram that the degradation is slower in the pH 1.0 hydrochloric acid medium, and the material conservation is between 90 - 110%, with mass conservation; In the pH 4.5 acetate medium and water medium, both degrade to carbidopa impurity G, and the material conservation is between 90 - 110%, with mass conservation; In the pH 6.8 phosphate medium, it degrades to carbidopa impurity G and α-methyl-3,4-dihydroxyphenylpropionic acid, and the material conservation is between 90 - 110%, with mass conservation.

[0075] Example 2

[0076] A detection method for the dissolution of levodopa and carbidopa and their degradation impurities in levodopa and carbidopa bulk drugs or preparations by HPLC

[0077] Step 1: Preparation of carbidopa stock solution: Weigh about 12 mg of carbidopa and place it in a 50-ml volumetric flask. Dissolve it with 0.1 mol / L hydrochloric acid solution and dilute to the mark to obtain the solution.

[0078] Step 2: Preparation of carbidopa linear solutions, as shown in the following table:

[0079] Table 2 Preparation of carbidopa linear solutions

[0080]

[0081] Step 3: Preparation of carbidopa impurity G stock solution: Weigh about 12 mg of carbidopa impurity G and place it in a 100-ml volumetric flask. Dissolve it with 0.1 mol / L hydrochloric acid solution and dilute to the mark to obtain the solution.

[0082] Step 4: Preparation of carbidopa impurity G linear solutions, as shown in the following table:

[0083] Table 3 Preparation of carbidopa impurity G linear solutions

[0084]

[0085] Step 5: Preparation of α-methyl-3,4-dihydroxyphenylpropionic acid stock solution: Weigh about 12 mg of α-methyl-3,4-dihydroxyphenylpropionic acid and place it in a 100-ml volumetric flask. Dissolve it with 0.1 mol / L hydrochloric acid solution and dilute to the mark to obtain the solution.

[0086] Step 6: Preparation of α-methyl-3,4-dihydroxyphenylpropionic acid linear solutions, as shown in the following table:

[0087] Table 4 Preparation of α-methyl-3,4-dihydroxyphenylpropionic acid linear solutions

[0088]

[0089] Step 7: Chromatographic conditions: Use octadecylsilane chemically bonded silica gel as the filler (Waters XBridge C18, 4.6×250 mm, 5 μm or a chromatographic column with equivalent column efficiency); use 0.1% trifluoroacetic acid solution - ethanol (90:10) as the mobile phase; the flow rate is 1.0 ml per minute; the column temperature is 30 °C; the sample tray temperature is 6 °C; the detection wavelength is 280 nm; the injection volume is 20 μl.

[0090] The above linear solutions are all prepared with pH 4.5 acetate solution: 0.1 mol / L hydrochloric acid solution (50:50) as the solvent.

[0091] Conclusion:

[0092] Table 5 Linear experiment results of carbidopa in pH 4.5 acetate buffer solution

[0093]

[0094] Table 6 Linear experiment results of carbidopa impurity G in pH 4.5 acetate buffer solution

[0095]

[0096] Table 7 Linear experiment results of α-methyl-3,4-dihydroxyphenylpropionic acid in pH 4.5 acetate buffer solution

[0097]

[0098] Example 3

[0099] An HPLC method for determining the dissolution of levodopa and carbidopa in levodopa and carbidopa raw materials or preparations. In the specific steps, the chromatographic column is packed with octadecylsilane-bonded silica gel (Waters XBridge C18 4.6*250mm, 5μm), the dissolution medium is pH 6.8 phosphate buffer solution, and the rest is the same as in Example 2:

[0100] Conclusion:

[0101] Table 8 Linear experiment results of carbidopa in pH 6.8 phosphate buffer solution

[0102]

[0103] Table 9 Linear experiment results of carbidopa impurity G in pH 6.8 phosphate buffer solution

[0104]

[0105] Table 10 Linear results of α-methyl-3,4-dihydroxyphenylpropionic acid in pH 6.8 phosphate buffer solution

[0106]

[0107] Example 4

[0108] An HPLC method for determining the dissolution of levodopa and carbidopa in levodopa and carbidopa raw materials or preparations. In the specific steps, the chromatographic column is packed with octadecylsilane-bonded silica gel (Waters XBridge C18 4.6*250mm, 5μm), the dissolution medium is water, and the rest is the same as in Example 2:

[0109] Table 11 Linear results of carbidopa in water medium

[0110]

[0111] Table 12 Linear Results of Carbidopa Impurity G in Aqueous Medium

[0112]

[0113] Table 13 Linear Results of α-Methyl-3,4-Dihydroxyphenylpropionic Acid in Aqueous Medium

[0114]

[0115]

[0116] Example 5

[0117] An HPLC detection method for determining the dissolution of levodopa, carbidopa and their degradation impurities in levodopa and carbidopa bulk drugs or preparations. The calibration factor results of the degradation impurities are shown in the following table:

[0118] Table 14 Calibration Factor Results of Degradation Impurities

[0119]

[0120] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device.

[0121] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Dissolution detection method for levodopa and carbidopa degradation impurities in controlled-release carbidopa-levodopa tablets, characterized in that, It includes the following steps: Step 1: Use liquid chromatography for detection. Using a specific chromatographic column for gradient elution has high separation efficiency; Step 2: The mobile phase is composed of ethanol and trifluoroacetic acid solution in a specific ratio; Step 3: Take an appropriate amount of Carbidopa and Levodopa Sustained Release Tablets, conduct a dissolution curve experiment. Take the dissolution solutions at each time point, filter, discard 1 ml, and take the subsequent filtrate: Dilute it with 0.1 mol / L hydrochloric acid solution (50:50) as the test solution; Step 4: Take appropriate amounts of levodopa and carbidopa reference substances, accurately weigh them, dissolve and dilute with 0.1 mol / L hydrochloric acid to prepare a solution containing about 0.03 mg of carbidopa and about 0.11 mg of levodopa per 1 ml as the reference solution; Step 5: Obtain the chromatograms of the test solution and the reference solution under qualified chromatographic separation conditions, and determine the dissolution of levodopa and carbidopa by the external standard method.

2. The dissolution detection method for levodopa and carbidopa degradation impurities in the controlled-release carbidopa and levodopa tablets according to claim 1, characterized in that: The chromatographic column for the liquid chromatography detection is packed with octadecylsilane chemically bonded silica gel.

3. The dissolution detection method for the degradation impurities of levodopa and carbidopa in the controlled-release tablet of carbidopa and levodopa according to claim 1, wherein: The mobile phase for the liquid chromatography detection includes mobile phase A and mobile phase B. Mobile phase A is 0.1% trifluoroacetic acid solution; mobile phase B is ethanol. Among them, the concentration of the trifluoroacetic acid solution can be 0.005%, 0.05%, 0.2% or 0.5%, etc., but is not limited to the values listed above. Other unlisted values within the above value range are equally applicable.

4. The dissolution detection method for the degradation impurities of levodopa and carbidopa in the controlled-release carbidopa and levodopa tablets according to claim 1, wherein: The flow rate of the mobile phase for the liquid chromatography detection is 0.8 - 1.2 ml / min, and the column temperature is 25 - 45°C. Among them, the flow rate of the mobile phase can be 0.8 ml / min, 0.9 ml / min, 1.0 ml / min, 1.1 ml / min or 1.2 ml / min, etc., and the column temperature can be 25°C, 30°C, 35°C, 40°C or 45°C, etc., but is not limited to the values listed above. Other unlisted values within the above value range are equally applicable.

5. The dissolution detection method for the degradation impurities of levodopa and carbidopa in the controlled-release tablet of carbidopa and levodopa according to claim 1, characterized in that: The detection wavelength for the liquid chromatography detection is 260 - 300 nm.

6. The dissolution detection method for the degradation impurities of levodopa and carbidopa in the controlled-release carbidopa and levodopa tablets according to claim 1, wherein: The elution program for the liquid chromatography detection is isocratic elution, mobile phase A: mobile phase B - 90:10, but is not limited to the values listed above. Other unlisted values within the above value range are equally applicable.

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