JPS6258166A - Method for simultaneous quantitative analysis of water-soluble vitamins in mixed vitamin preparation - Google Patents

Abstract

PURPOSE:To enable the quantification of water-soluble vitamins in a mixed vitamin preparation by one analysis, by subjecting the mixed vitamin preparation to an ion-pair chromatograph using a mobile phase consisting of a polar solvent, a phosphate buffer solution and an ion-pair reagent and subsequently analyzing the eluate by a fluorescent method using o-phthalaldehyde as a fluorescent reagent. CONSTITUTION:The simultaneous quantitative analysis of water-soluble vitamins in a mixed vitamin preparation can be rapidly performed with high accuracy by combining an ion-pair chromatograph method using a mobile phase consisting of a polar solvent, a phosphate buffer solution and an ion-pair reagent with a pantenol detection method according to an ortho-phthalaldehyde fluorescent method optimum to said mobile phase. As the polar solvent, methanol is especially pref. and, as the phosphate buffer solution, one with a phosphate-potassium concn. of 0.01-0.02M and pH3-3.5 is especially pref. As the ion-pair reagent, sodium pentasulfonate is especially pref.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for simultaneous determination of water-soluble vitamins in mixed vitamin preparations.

In this specification, water-soluble vitamins refer to vitamins such as ascorbic acid, thiamine hydrochloride, sodium riboflavin phosphate, pyridoxine hydrochloride, nicotinamide, and no-Q-totenol.

[Conventional technology]

Conventionally, the quantitative determination of vitamins in mixed vitamin preparations has been carried out by manual analysis using colorimetric methods, UV methods, titration methods, microbiological quantitative methods, etc. for each individual vitamin, which requires a lot of labor and time. It was work.

However, in conjunction with recent technical advances in high-performance liquid chromatography, many methods for the simultaneous determination of water-soluble vitamins using high-performance liquid chromatography have been reported within the past few years [Sharnal of Journal of Pharmac
euticalScience) 70, 1014
~1017 (198i L same, 70.90~101 (
1981); 67° 1444-1446 (1978
)). These methods can be broadly divided into methods that use ion-exchange resins as fillers and separate by ion-exchange action, and reversed-phase chromatography methods that use non-polar fillers.

However, in recent years, the latter reverse phase has been solved using the Madgraph method, especially,
Add appropriate opposing ions to the mobile phase to form ion pairs,
Ion-pair chromatography, which uses a non-polar stationary phase for separation, has become popular.

[The problem that the invention is trying to solve]

However, these simultaneous assay methods cannot simultaneously quantify all of the water-soluble vitamins mentioned above. That is, there have been no reports of separating and quantifying sodium riboflavin phosphate, which is frequently used in water-soluble pharmaceuticals, from impurities and decomposition products derived from it, as well as thiamine hydrochloride, pyridoxine hydrochloride, ascorbic acid, and nicotinic acid amide. In addition, regarding NoQ-totenol, its UV absorption is at a wavelength of 200 nm.
Because it is just nearby and there is no absorption at all other than that,
Simultaneous determination of 6111 with other water-soluble vitamins was difficult.

[Means for solving problems]

Under such circumstances, the present inventors conducted extensive research and found that the ion pair O? ) by graphical methods and optimal orthophthalaldehyde fluorescence for this mobile phase. By combining the e-totenol detection method, the present invention was completed by discovering that the above water-soluble vitamins can be simultaneously and simultaneously quantified in L quickly and with high precision.

That is, in the present invention, a mixed vitamin preparation is subjected to ion pair chromatography using a mobile phase consisting of the following ■ - ■, ■ polar solvent ■ phosphate buffer ■ ion pair reagent, and then the eluent is treated with a fluorescent reagent. The present invention provides a method for simultaneous determination of water-soluble vitamins in a mixed vitamin curing agent, which is characterized by analysis by a fluorescence method using ortho-7-talaldehyde.

Hereinafter, the method of the present invention will be explained with reference to FIG. 1, which shows an example of an analyzer used therein.

The analyzer includes an ion pair chromatograph indicated by A;
It consists of a no-Q totenol detection system indicated by B connected in series to this. The ion pair chromatograph A consists of a column indicated by 1, a UV detector indicated by 2, a mobile phase indicated by 3, etc., and has the same configuration as a normal chromatograph.

In addition, the NoQ totenol detection system B includes an OPA fluorescence system shown as B-1 for performing a fluorescence method using ortho-phthalaldehyde as a fluorescence reagent (hereinafter referred to as OPA fluorescence method), and B- It consists of a fluorescence detector shown as 2. OPA fluorescence system B-1 is 3
Two reaction tubes are connected in series to detect non-9totenol using the OPA fluorescence method.

Chromatographic operations using ion pair chromatograph A can be carried out according to conventional methods. Examples of the column 1 include those obtained by treating a silica gel carrier with octadecylsilane, such as TSK-GeJ 0DS-120TC (manufactured by Toyo Soda Kogyo), μm bonder, eTsuku C1m (manufactured by Waters, USA), etc. Especially TSK-Gej
0DS-120T is preferred.

As the mobile phase 3, a solution consisting of a phosphate buffer, a polar solvent, and an ion pairing reagent is used. As a polar solvent,
Examples include ethanol and methanol, with methanol being particularly preferred. Phosphate buffer is made by adding potassium phosphate to water and adjusting the pH with phosphoric acid, and the concentration of potassium phosphate is 0.01 to 0.02M, pH
is particularly preferable. The mixing ratio of phosphate buffer and polar solvent is generally 80:20 to 90:1.
The ratio is adjusted to 0, but when the polar solvent is methanol, the ratio is particularly preferably 84:16.

Further, examples of the ion pairing reagent include sodium pentanesulfonate, sodium hexanesulfonate, sodium hexanesulfonate, etc. Among them, sodium pentanesulfonate is particularly preferred. The ion-pairing reagent is KO, 05-0.2 w/v (%) in the total composition of the mobile phase.
) is preferable, and good separation cannot be obtained outside this range.

Note that the mobile phase is normally delivered at a flow rate of about 1.0 m/min.

Moreover, it is preferable that the UV detector 2 performs detection at a wavelength of 270 to 280 nm.

Thus, perform a Madograph analysis of ions, ascorbic acid, nicotinamide, salt! Pyridoxine, non-9totenol, thiamine hydrochloride, and sugoflavin sodium phosphate are eluted in this order. However, NoQ-totenol cannot be detected here because it has no UV absorption at 270 to 280 nm, and is therefore detected using the OPA fluorescence method. Q-totenol detection system B makes it possible to quantify it for the first time.

OPA fluorescence system B-1 has three reaction tubes,
In reaction tube a, add a certain concentration of alkaline solution to the eluent,
Upon heating, Qtotenol is hydrolyzed to β-alanine. In the reaction tube, after hydrolysis, an acid is added to neutralize the liquid, and a fluorescent reagent containing orthophthalaldehyde is added to the syneresis to generate a fluorescent substance. Quantify with

As the alkali M7&, an aqueous thorium hydroxide solution is preferable, the concentration is 1 to 2 normal, and the concentration is about 0.1me1 minute. Preferably added at j&t.

Further, the acid is added at the same concentration and at the same flow rate as the alkaline solution used. The hydrolysis temperature is preferably 80 to 90°C; if it is higher than this, for example, if methanol is used as the mobile phase solvent, bubbles will be generated and the analysis accuracy will be reduced. A pH 9.0 borate buffer containing % orthophthalaldehyde is preferred, and the reaction temperature is preferably about 50° C. The emission light is detected at an excitation wavelength of 345 nm.

It is preferable to carry out the detection at a wavelength of 455 nm.

Note that the fluorescent reagent solution is normally fed at a flow rate of about 1.0 rnl/min.

[Effect]

The mobile phase used in the present invention satisfactorily elutes water-based vitamins and their derived impurities and decomposition products, and derivatizes orthophthalaldehyde into a fluorescent substance in the eluent. be able to.

〔Effect of the invention〕

Since the present invention is a paper-based method, water-soluble vitamins such as thiamin hydrochloride, phosphorus & IJ boflavin sodium, ascorbic acid, pyridoxine hydrochloride, nicotinamide, and noQ-totenol in the preparation can be determined in one analysis. It is possible to quantify. With conventional high-performance liquid chromatography technology, only three of these can be quantified simultaneously.
There was a limit to the extent of the components, and other components had to be tested individually using fluorescence methods, titration methods, colorimetric methods, etc. Therefore,
According to the method of the present invention, it is possible to perform quantitative analysis with a reliability rate of 98% or more in terms of accuracy and the operation time is within one hour, making the present invention very effective in pharmaceutical analysis.

〔Example〕

Next, an example will be given and explained.

Example 1 Water-soluble vitamins in a mixed vitamin preparation for infusion were determined by the following method.

U) Procedure Ascorbic acid 1001f, Nicotinic acid amide 20■,
3 grams of pyridoxine hydrochloride, 10 grams of thiamine hydrochloride, 2 grams of sodium riboflavin phosphate 1. Qnttail 5119
Accurately weigh a sample (1ffi/crystal) corresponding to , add the mobile phase and precisely 100-mm (hereinafter referred to as standard solution), apply it to a liquid chromatograph under the following conditions, and simultaneously quantify the above six components. .

Perform the same procedure for the infusion vitamin mixture,
Compare with the results of the standard solution.

■) Separation conditions Column conditions: TSK-Gel ODS-120
T column tube: inner diameter 41, length 25 m Mobile phase: 0.01M phosphate-potassium solution (pH 3
, 5) Methanol (250:50) + 0.1% Sodium 1-pentanesulfonate elution rate: 1.0Wt1. /min Detection wavelength: UV277nm Column temperature: room temperature Injection amount = 20μ! (3) Equipment used: Shimadzu LC-3A high performance liquid chromatograph (4) NoQ totenol detection conditions ■ Hydrolyzed alkaline solution = 2N-sodium hydroxide flow rate: 0.
1-/min Reaction temperature: 90℃ ■ Neutralizing acid: 2N-hydrochloric acid Flow rate: 0.1 m/min Reaction temperature: Room temperature ■ Issuing photoreaction photovoltaic test solution: 0.05% OPA, 0.2% 2-
Mercaptoethanol, pH 9.0 borate buffer containing 10% ethanol Flow rate: 1. Ord/min Reaction temperature: 50°C 6) Sample (injection) The sample used had the following formulation.

(Prescription) Vitamin A 10,000IU ergocalciferol 1,000IU tocopherol acetate 5-pound thiamine hydrochloride
50 Shoulder 2 Phosphate Frapine Sodium
10mp Pyridoxine Hydrochloride 15 Nicotinamide 100■NoQ Totenol 25119 Ascorbic Acid
5008F! 57 with distilled water for injection! shall be.

(6) The peak of each component detected by the method of the present invention is the first
As shown in the figure.

Example 2 Water-soluble vitamins in internally administered vitamin preparations were determined by the following method.

(1) Procedure: Accurately weigh samples (crystal 20-) corresponding to nicotinic acid amide 15, 119 thiamine hydrochloride, sugoflavine sodium phosphate 2, and 3011 g of non-9totenyl alcohol, and add the mobile phase. The solution was subjected to liquid chromatography under the following conditions to simultaneously quantify the above four components.

ri) Separation φ items Same as Example 1.

(3) Use @ observation Same as Example 1.

(4) NoQ totenol detection conditions Same as Example 1.

(!19 Sample (liquid) The sample used had the following formulation.

(Prescription) Keishi Toryu Extract 50,000 Ginseng Kiss 50. +9 Thiamine hydrochloride
10.9 Rizeflavin Sodium Phosphate
2-rich 9-methyl hesubericin
1 (1g nicotinic acid amide 15+g/
Q Totenol 30+q Zoyakochi/Ki 0.05m Goou tincture
20rn1. with 0.05g purified water. shall be.

(6) The peaks of each component detected by the method of the present invention are shown in FIG.

[Brief explanation of drawings]

Figure 1 is a chromatogram when a mixed vitamin preparation for human use was analyzed using the method of the present invention, Figure 2 is a chromatogram when an oral vitamin preparation was analyzed using the method of the present invention, and Figure 3 is a chromatogram when a vitamin preparation for oral use was analyzed using the method of the present invention. It is a schematic explanatory diagram showing an example of the analysis device used. Above is Figure 1

Claims (1)

[Scope of Claims] 1. A mixed vitamin preparation according to the following (1) to (3), (1)
It is subjected to ion pair chromatography using a mobile phase consisting of a polar solvent (2) phosphate buffer (3) ion pairing reagent, and then the eluent is analyzed by a fluorescence method using orthophthalaldehyde as a fluorescence reagent. A method for simultaneous determination of water-soluble vitamins in a mixed vitamin preparation, characterized by: