A method for determining the content of multiple indicators of Jinrong granules in blood

Abstract

This invention discloses a method for determining the content of multiple indicators of Jinrong granules absorbed into the bloodstream. The ion chromatogram is obtained using UPLC-Q-TOF-MS / MS analysis. The chromatographic conditions include: flow rate: 0.3–0.5 mL / min; column temperature: 20–30℃; mobile phase: phase A is acetonitrile, and phase B is a 0.04–0.08 mmol / L ammonium formate aqueous solution. The mobile phase comprises acetonitrile and a 0.04–0.08 mmol / L ammonium formate aqueous solution. This invention can simultaneously determine the content of multiple major components of Jinrong granules after absorption into the bloodstream, enabling more comprehensive control of the quality of Jinrong granules. It provides a convenient and efficient method for determining the content of components both in vivo and in vitro, laying a scientific foundation for improving the overall quality standards of Jinrong granules.

Description

Technical Field

[0001] This invention relates to methods for determining the content of drug components, and in particular to a method for determining the content of multiple indicators of components entering the bloodstream from Jinrong granules. Background Technology

[0002] Jinrong Granules are an innovative traditional Chinese medicine for the treatment of breast hyperplasia. On April 12, 2005, it received clinical approval from the State Food and Drug Administration as "Xiaopi Granules" (2005L01119), and in December 2018, it was approved for marketing as an innovative traditional Chinese medicine "Jinrong Granules" (National Drug Approval Number Z20180002).

[0003] Jinrong Granules are composed of Epimedium, Cistanche deserticola, Curcuma longa, Salvia miltiorrhiza, Leonurus japonicus, Curcuma zedoaria, processed Polygonum multiflorum, Ligustrum lucidum, turtle shell, and oyster shell. Its functions and indications are to tonify the kidney and activate blood circulation, resolve phlegm and dissipate nodules, and regulate the Chong and Ren meridians. It is used for breast hyperplasia with phlegm and blood stasis and Chong and Ren meridian imbalance, with symptoms such as breast pain and tenderness, chest and rib pain, depression and irritability, insomnia and dreaminess, fatigue, weakness in the lower back and knees, pale red or bluish-purple tongue or ecchymosis on the tongue edge and tip, white tongue coating, and wiry and thready or slippery pulse.

[0004] Accordingly, the main characteristic components detected in this invention are: icariin, cymosiderin I, and magnoflorine, which are the pharmacopoeia-specified index components and main active components of Epimedium; echinacoside, which is the pharmacopoeia-specified index component and main active component of Cistanche deserticola; tanshinone I, tanshinone IIA, and cryptotanshinone, which are the pharmacopoeia-specified index components and main active components of Salvia miltiorrhiza; and stachydrine, which is the pharmacopoeia-specified index component and main active component of Leonurus japonicus.

[0005] The determination of echinacoside and verbascoside in Jinrong Xiaopi Granules by HPLC method [J]. Citation Edition: Medicine and Health, 2015, 1(4): 20-20. The article “Zhou Ruopeng, Deng Xiaojun. Determination of echinacoside and verbascoside in Jinrong Xiaopi Granules by HPLC method [J]. Citation Edition: Medicine and Health, 2015, 1(4): 20-20” reports on the determination of echinacoside and verbascoside in Jinrong Granules. The article “Zhou Ruopeng. Establishment of fingerprint spectrum of Jinrong Xiaopi Granules, 2015” reports on the fingerprint spectrum study of Jinrong Granules. The article identifies four common peaks in Jinrong Granules: echinacoside, verbascoside, salvianolic acid B, and icariin, and assigns these peaks to medicinal materials. However, it does not quantitatively analyze the characteristic components representing the dose-effect relationship in the prescription. Jinrong Granules are prepared according to the “National Drug Administration Standard (YBZ00042018)” for the determination of icariin content. However, this single-index quantification is insufficient to truly and comprehensively control the intrinsic quality of the preparation.

[0006] Therefore, given the current quality control issues of Jinrong Granules, there is an urgent need to invent analytical methods that can detect representative components of Jinrong Granules. Based on this, qualitative analytical methods for absorbed components and methods for determining the content of major components should be established. This will preliminarily elucidate the pharmacodynamic material basis of Jinrong Granules and provide research methods to solve the problem of difficult monitoring of trace components entering the blood. This has important scientific significance and practical value for improving the quality control of Jinrong Granules. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for determining the content of multiple indicators of Jinrong granules in the blood, which addresses the shortcomings of the existing technology. This method can simultaneously determine the content of multiple main components of Jinrong granules after absorption into the blood, and can more comprehensively control the quality of Jinrong granules.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0009] A method for determining the content of multiple indicators of Jinrong granules entering the blood, using UPLC-Q-TOF-MS / MS analysis to obtain the ion chromatogram;

[0010] The chromatographic conditions include:

[0011] Column: C18

[0012] Flow rate: 0.3–0.5 mL / min;

[0013] Column temperature: 20~30℃;

[0014] Mobile phase: Phase A is acetonitrile, and Phase B is an aqueous solution of 0.04–0.08 mmol / L ammonium formate. The mobile phase includes acetonitrile and an aqueous solution of 0.04–0.08 mmol / L ammonium formate.

[0015] The following elution procedure was used for elution:

[0016] 0–10 min: 5% → 15% flow phase A, 95% → 85% flow phase B;

[0017] 10–15 min: 15% → 35% flow phase A, 85% → 65% flow phase B;

[0018] 15–25 min: 35% → 65% flow phase A, 65% → 35% flow phase B;

[0019] 25–40 min: 65% → 95% flow phase A, 35% → 5% flow phase B.

[0020] This invention utilizes ultra-high performance liquid chromatography (UHPLC) for higher resolution and shorter analysis time. When coupled with mass spectrometry, it can obtain information on chemical components without UV absorption. Furthermore, through molecular feature extraction, it can identify chemical components in the drug serum that are masked by protein ion peaks, enabling rapid and efficient determination of the types and contents of components absorbed into the bloodstream by Jinrong granules.

[0021] Jinrong granules have a complex composition, including flavonoids and their glycosides, alkaloids, phenolic acids, and phenylethanoid glycosides. To achieve better separation of the components, a gradient elution with decreasing polarity was used. The total elution time was set based on the peak characteristics, and the baseline became relatively smooth after 40 minutes of elution. The mobile phase B was an ammonium formate aqueous solution. During the first 10-15 minutes of elution, phase B was dominant, and the mobile phase was strongly acidic, preferentially separating alkaloids. During the last 10-15 minutes, phase A became dominant, and the acidity of the mobile phase decreased, allowing the detection of acidic phenolic acid components. The formulation contains a large number of glycosides. Between 15-25 minutes, the mobile phase was neutral to low polarity. As the polarity of the mobile phase decreased, polysaccharides, disaccharides, and aglycones eluted sequentially.

[0022] The purpose of adding ammonium formate to the aqueous phase is to adjust the pH value of the solution. Ammonium formate has good solubility, is fully compatible with the liquid chromatography-mass spectrometry method, and causes relatively little damage to the chromatographic column.

[0023] The UPLC-Q-TOF-MS / MS analytical method refers to Ultra Performance Liquid Chromatography-Quadrupole-Time of Flight-Mass Spectrometry.

[0024] In a preferred embodiment of the present invention, the mass spectrometry detection conditions are as follows: the analysis mode is positive and negative ion mode, ESI ionization mode, and Agilent standard tuning solution ESI-L Low Concentration TuningMix (G1969-85000) is used to perform accurate mass number calibration before sample injection analysis;

[0025] Detection range of primary mass spectrometry scanning: m / z 100~1700;

[0026] Solvent-removing drying gas: nitrogen;

[0027] Temperature: 325℃;

[0028] Flow rate: 6.8 L / min;

[0029] Sheath gas temperature: 350℃;

[0030] Capillary voltage: 4.0 kV;

[0031] Fragment voltage: 150V.

[0032] In a preferred embodiment of the present invention, the serum sample solution of Jinrong granules is used as the sample solution in the UPLC-Q-TOF-MS / MS analysis method.

[0033] In a preferred embodiment of the present invention, the method for preparing the Jinrong granule drug serum sample solution includes weighing and dissolving Jinrong granules, administering the drug to rats by gavage, collecting blood, separating serum, precipitating proteins, centrifuging, concentrating, reconstituted, and filtering to obtain the solution.

[0034] In a preferred embodiment of the present invention, the dosage of the Jinrong granules is 2 to 4 times the clinically equivalent dose.

[0035] The clinically equivalent dose is the relative drug concentration or dose that can elicit an equivalent response. It reflects the relationship between drug effect and drug dose. The clinically equivalent dose is 0.8–1.2 g.

[0036] In a preferred embodiment of the present invention, the rats are administered the drug twice a day for 7 consecutive days.

[0037] In a preferred embodiment of the present invention, the specific method after separating the serum is as follows: take 2 ml of serum, precipitate the protein with methanol, centrifuge, concentrate, reconstitute, and filter through a 0.22 μm filter membrane.

[0038] In a preferred embodiment of the present invention, the chromatographic column has the following specifications: 3.0 × 100 mm, 1.8 μm.

[0039] In a preferred embodiment of the present invention, the mixed control blank serum solution in the UPLC-Q-TOF-MS / MS analysis method includes icariin, cymosin I, echinacoside, verbascoside, tanshinone I, tanshinone IIA, cryptotanshinone, ligustrum lucidum glycoside, magnoflorine, stachydrine, and salvianolic acid B.

[0040] In a preferred embodiment of the present invention, the mixed control blank serum solution is prepared by taking 2-3 ml of blank serum, adding 100 μl of mixed control solution, precipitating proteins with methanol, concentrating, reconstituted, and filtering through a filter membrane;

[0041] The concentrations of icariin, baicalin I, echinacoside, verbascoside, tanshinone I, tanshinone IIA, cryptotanshinone, ligustrazine, magnoflorine, stachydrine, and salvianolic acid B in the mixed reference solution were 0.4–0.5 mg / mL, 0.4–0.5 mg / mL, 0.5–0.6 mg / mL, 0.4–0.5 mg / mL, 0.4–0.5 mg / mL, 0.35–0.5 mg / mL, 0.35–0.5 mg / mL, 0.4–0.5 mg / mL, 0.4–0.5 mg / mL, 0.6–0.8 mg / mL, and 0.5–0.6 mg / mL, respectively.

[0042] In a preferred embodiment of the present invention, the method further includes the preparation of a sample solution of Jinrong granules: weigh 2-4g of dry extract powder of Jinrong granules, dissolve in methanol, sonicate, centrifuge, filter through a 0.22μm microporous membrane, and place in a sample vial.

[0043] The specific conditions for centrifugation, concentration, and ultrasonic treatment of the serum samples are as follows:

[0044] Protein removal centrifugation conditions: 5000 rpm, 10 min;

[0045] Ultrasonic conditions: 40-60 kHz, 15 min;

[0046] Reconstitution centrifugation conditions: 12000 rpm; 5 min.

[0047] The specific conditions for centrifugation and ultrasonic treatment of the Jinrong granule sample and the mixed reference solution are as follows:

[0048] Ultrasonic conditions: 40-60 kHz, 15 min;

[0049] Centrifugation conditions: 8000 rpm; 5 min.

[0050] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0051] 1. The determination method provided by this invention uses ultra-high performance liquid chromatography (UHPLC) with higher resolution and shorter analysis time. When coupled with mass spectrometry, it can obtain information on chemical components without ultraviolet absorption. Furthermore, through molecular feature extraction, it can obtain chemical components in drug serum that are masked by protein ion peaks, and rapidly and efficiently determine the type and content of components absorbed into the blood by Jinrong granules.

[0052] 2. The method for determining the content of multiple components entering the blood provided by the present invention, wherein the standard component preparation has the corresponding components in Jinrong granules, and the determination is carried out by UPLC-Q-TOF-MS method to simultaneously detect multiple indicator components in the same spectrum;

[0053] 3. Detecting the components of Jinrong granules absorbed into the bloodstream will help in the basic research on the efficacy of Jinrong granules, realize the basic quality evaluation of the efficacy of Jinrong granules, and effectively control the quality of Jinrong granules. Attached Figure Description

[0054] Figures 1a-1d These are the total ion chromatograms of the Jinrong granule sample solution and the Jinrong granule drug serum sample solution provided in Example 1 of this invention.

[0055] Figures 2a-2b This is the total ion chromatogram of the mixed reference solution in Example 1 provided by the present invention;

[0056] Figures 3a-3b These are the ion current extraction chromatograms of the standard components in the *Cistanche deserticola* granule sample solution and the total ion current integral chromatogram of the mixed reference solution provided in Example 1 of this invention.

[0057] Figure 4a These are the ion current extraction diagrams of the standard components and the mixed control blank serum solution in the serum sample solution of Jinrong granules provided in Example 1 of this invention.

[0058] The standard ingredients include: icariin, magnoflorine, cynomolgin I, echinacoside, stachydrine, tanshinone I, tanshinone IIA, and cryptotanshinone. Detailed Implementation

[0059] The present invention will be further described in detail with reference to the accompanying drawings and embodiments. The specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0060] 1. Instruments and equipment: Agilent 1290 / 6540UPLC-Q-TOF / MS chromatography-mass spectrometry system; Agilent ZORBAX Eclipse Plus C18 (3.0×100mm, 1.8μm); ultrasonic instrument; S-23 high-speed benchtop centrifuge; AUW120D electronic analytical balance; vacuum centrifuge concentrator.

[0061] Reference standards, i.e., standard component preparations, include: stachydrine hydrochloride (content 98.81%), rhodioloside (content 99.77%), magnoflorin (content 99.01%), echinacoside (content 98.98%), verbascoside (content greater than 98%), privetin (content 99.52%), salvianolic acid B (content 94.53%), asparagine C (content 98.19%), icariin (content 98.47%), baicalin I (content 97.75%), emodin (content 98.83%), cryptotanshinone (content 98.79%), tanshinone I (content 99.37%), and tanshinone IIA (content 99.53%).

[0062] Reagents and materials: methanol (chromatographic grade), acetonitrile (mass spectrometry grade), ammonium formate (mass spectrometry grade), distilled water.

[0063] 2. Source of drugs: Jinrong Granules Dry Paste (batch number: 170801-1) and Volatile Oil Component Inclusion Complex (batch number: B-170807) were provided by Guangzhou Qiji Pharmaceutical Technology Co., Ltd.

[0064] Example 1

[0065] Step 1, Jinrong Granules Sample Solution: Weigh about 3g of dry extract powder of Jinrong granules, add 30ml of methanol solution, sonicate for 30min, let stand, take 2ml of solution and centrifuge (8000rpm, centrifuge for 5min), filter the supernatant through a 0.22μm microporous membrane, and transfer it to a sample vial.

[0066] Step 2: Mixing the reference solutions: Accurately determine 10 mg of the reference standard in a 25 ml volumetric flask, add 15 ml of methanol, sonicate to dissolve, shake well, and dilute to volume to obtain the reference solution. Take 100 μl of each reference solution, mix well, filter through a 0.22 μm filter membrane, and transfer to a sample vial. The concentrations of each characteristic component in the reference solution should be: stachydrine 14.43 μg / ml, magnoflorine 31.65 μg / ml, echinacoside 11.08 μg / ml, verbascoside 4.60 μg / ml, privetin 5.28 μg / ml, icariin 15.43 μg / ml, baicalin I 33.33 μg / ml, cryptotanshinone 38.52 μg / ml, tanshinone I 28.68 μg / ml, and tanshinone IIA 47.12 μg / ml.

[0067] Step 3: Serum Preparation: Twenty female SPF-grade SD rats were randomly and equally divided into a control group and a Jinrong granule group. The rats were labeled and their body weight was recorded. The 20 rats underwent acclimatization culture for 3 days. On day 4, they were administered the granules via gavage (calculated based on the equivalent dose ratio) for 7 consecutive days. Before the last administration, the rats were fasted for 12 hours but allowed free water. One hour after gavage, the rats were anesthetized by intraperitoneal injection of ketamine-xylazine (60 mg / kg(K) + 10 mg / kg(X)). Blood was collected from the abdominal aorta after anesthesia and centrifuged at 3000 rpm, 4℃, for 5 minutes. The supernatant was collected and filtered through a 0.22 μm filter in a laminar flow hood. The filtrate was transferred to sterile centrifuge tubes. The serum was labeled according to the animal group and stored frozen.

[0068] Step 4, Preparation of Serum Sample Solutions: ① Preparation of Jinrong Granules Serum Sample Solution: Weigh three times the clinically equivalent dose of Jinrong Granules, dissolve them, and administer to rats by gavage for 7 days, twice a day. Collect blood and separate serum. Take 2 ml of serum, precipitate proteins with methanol, centrifuge, concentrate, reconstitute, filter through a 0.22 μm filter membrane, and place in a sample bottle. ② Preparation of Mixed Control Blank Serum Solution: Take 2 ml of blank serum, add 100 μl of the mixed control solution described in Step 2, precipitate proteins with methanol, concentrate, reconstitute, filter through a 0.22 μm filter membrane, and place in a sample bottle.

[0069] Step 5: Detect the content of the reference solution and the test solution using UPLC-Q-TOF-MS / MS method;

[0070] Chromatographic conditions during the detection process:

[0071] Column: Agilent ZORBAX Eclipse Plus C18 (3.0×100mm, 1.8μm)

[0072] Mobile phase composition: Phase A: Acetonitrile (organic phase); Phase B: 0.05 mmol / L ammonium formate aqueous solution (aqueous phase).

[0073] Injection volume: 1 μL

[0074] Flow rate: 0.4 mL / min

[0075] Gradient elution program selection: Jinrong granules have a complex composition, containing flavonoids and their glycosides, alkaloids, phenolic acids, and phenylethyl glycosides. To achieve better separation of the components, a gradient elution with decreasing polarity was used. The total elution time was set based on the peak elution characteristics, and the baseline became relatively smooth after 40 minutes of elution. The mobile phase B was an ammonium formate aqueous solution. During the first 10-15 minutes of elution, phase B predominated, and the mobile phase was strongly acidic, preferentially separating alkaloids. During the last 10-15 minutes, phase A predominated, and the acidity of the mobile phase decreased, allowing the detection of acidic phenolic acid components. The formulation contains a large number of glycosides. Between 15-25 minutes, the mobile phase was neutral to low polarity. As the polarity of the mobile phase decreased, polysaccharides, disaccharides, and aglycones eluted sequentially.

[0076] Gradient elution program settings: 0–10 min, 5%–15% A; 10–15 min, 15%–35% A; 15–25 min, 35%–65% A; 25–40 min, 65%–95% A

[0077] Mass spectrometry detection conditions:

[0078] Analysis Mode: Positive and Negative Ion Analysis Mode

[0079] Ionization method: Electrospray ionization

[0080] Accurate mass calibration solution before sample injection analysis: Agilent Standard Tuning Mix ESI-L Low Concentration Tuning Mix (G1969-85000)

[0081] Detection range of primary mass spectrometry scanning: m / z 100~1700;

[0082] Solvent-removing drying gas: nitrogen;

[0083] Temperature: 325℃;

[0084] Flow rate: 6.8 L / min;

[0085] Sheath gas temperature: 350℃;

[0086] Capillary voltage: 4.0 kV;

[0087] Fragment voltage: 150V

[0088] See Figures 1a-3b This is the chromatogram of the *Cistanche deserticola* granule sample solution in this embodiment.

[0089] Calculate the content of 10 components in the test solution: Calculate the content of stachydrine, icariin, cymosin I, echinacoside, verbascoside, tanshinone I, tanshinone IIA, cryptotanshinone, ligustrum lucidum glycoside, and magnoflorine in the test solution using the standard curve method.

[0090] The calculation results are shown in Table 1:

[0091] Table 1. Results of determination of the main components in Jinrong Granules

[0092]

[0093] See Figure 4a The image shows the chromatogram of the serum sample solution of Jinrong granules in this embodiment.

[0094] Based on the ion current extraction chromatograms of the serum sample solution of Jinrong granules and the blank serum solution of the mixed reference standard, and according to the concentration of the reference standard, the peak area of ​​the obtained reference standard and the peak area of ​​the corresponding standard component in the sample were detected, and the content of the corresponding standard component was calculated. The contents of the eight components in the test solution were calculated separately: the contents of stachydrine, icariin, cymosin I, echinacoside, tanshinone I, tanshinone IIA, cryptotanshinone, and magnoflorine in the test solution were calculated using the standard curve method. Calculation formulas: Cserum sample = Creference standard * Aserum sample / Creference standard; Content of each component in Jinrong granules = Cserum sample / CJinrong granule sample solution

[0095] Table 2. Results of determination of the main blood-entering components of Jinrong Granules

[0096]

[0097] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solution and inventive concept of the present invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A method for determining the content of multiple indicators of blood-entering components in Jinrong granules, characterized in that, Ion chromatograms were obtained using UPLC-Q-TOF-MS / MS analysis. Chromatographic conditions include: Column: C18 Flow rate: 0.3–0.5 mL / min; Column temperature: 20~30℃; Mobile phase: Phase A is acetonitrile, and Phase B is an aqueous solution of 0.04–0.08 mmol / L ammonium formate. The mobile phase includes acetonitrile and an aqueous solution of 0.04–0.08 mmol / L ammonium formate. The following elution procedure was used for elution: 0–10 min: 5% → 15% flow phase A, 95% → 85% flow phase B; 10–15 min: 15% → 35% flow phase A, 85% → 65% flow phase B; 15–25 min: 35% → 65% flow phase A, 65% → 35% flow phase B; 25–40 min: 65% → 95% mobile phase A, 35% → 5% mobile phase B; Mass spectrometry detection conditions: The analysis mode is positive and negative ion mode, ESI ionization mode, and Agilent standard tuning solution ESI-L Low Concentration Tuning Mix is ​​used to calibrate the accurate mass number before sample injection analysis; Detection range of primary mass spectrometry scanning: m / z 100~1700; Solvent-removing drying gas: nitrogen; Temperature: 325℃; Flow rate: 6.8 L / min; Sheath gas temperature: 350℃; Capillary voltage: 4.0 kV; Fragment voltage: 150V; In the UPLC-Q-TOF-MS / MS analysis method, the serum sample solution of Jinrong granules is used as the sample solution. The preparation method of the serum sample solution of Jinrong granules includes weighing and dissolving Jinrong granules, administering the drug to rats by gavage, collecting blood, separating serum, precipitating proteins, centrifuging, concentrating, reconstituted, and filtering to obtain the solution. The specific method after serum separation is as follows: take 2 ml of serum, precipitate proteins with methanol, centrifuge, concentrate, reconstitute, and filter through a 0.22 μm filter membrane; Column specifications: 3.0 × 100 mm, 1.8 μm; In the UPLC-Q-TOF-MS / MS analysis method, the mixed control blank serum solution includes icariin, cymosin I, echinacoside, verbascoside, tanshinone I, tanshinone IIA, cryptotanshinone, ligustrum lucidum glycoside, magnoflorine, stachydrine, and salvianolic acid B. Jinrong Granules are formulated with Epimedium, Cistanche deserticola, Curcuma longa, Salvia miltiorrhiza, Leonurus japonicus, Curcuma zedoaria, processed Polygonum multiflorum, Ligustrum lucidum, turtle shell, and oyster shell.

2. The method for determining the content of multiple indicators of blood-entering components of Jinrong granules according to claim 1, characterized in that, The dosage of the Jinrong granules is 2 to 4 times the clinically equivalent dose.

3. The method for determining the content of multiple indicators of blood-entering components of Jinrong granules according to claim 1, characterized in that, The rats were administered the drug twice a day for 7 consecutive days.

4. The method for determining the content of multiple indicators of blood-entering components of Jinrong granules according to claim 1, characterized in that, The method for preparing the mixed reference blank serum solution is as follows: take 2-3 ml of blank serum, add 100 μl of mixed reference solution, precipitate proteins with methanol, concentrate, reconstitute, and filter through a filter membrane; The concentrations of icariin, baicalin I, echinacoside, verbascoside, tanshinone I, tanshinone IIA, cryptotanshinone, ligustrazine, magnoflorine, stachydrine, and salvianolic acid B in the mixed reference solution were 0.4–0.5 mg / mL, 0.4–0.5 mg / mL, 0.5–0.6 mg / mL, 0.4–0.5 mg / mL, 0.4–0.5 mg / mL, 0.35–0.5 mg / mL, 0.35–0.5 mg / mL, 0.4–0.5 mg / mL, 0.4–0.5 mg / mL, 0.6–0.8 mg / mL, and 0.5–0.6 mg / mL, respectively.

Images