DE202005011957U1 - Salt solution, useful as ionization additives, comprises mixed organic acid components (e.g. formic acid) and base components (e.g. ammonia or methylamine) of the salt, where the acid content is high and water content is low - Google Patents

Abstract

Salt solution (I) in acetonitrile comprises mixed organic acid components and base components of the salt (both boils at normal pressure at less than 300[deg]C), where the amount of the acid component is up to 1 vol.% related to the total volume of acetonitrile and the water content is below 5%, determined by the Karl-Fischer titration. An independent claim is also included for the preparation of (I).

Description

The LC / MS, the coupling of liquid chromatography with mass spectrometry, since the 90s, the method the choice for various questions in the field of pharmaceutical, clinical and forensic chemistry, as well as in environmental analysis and food monitoring. Especially in the field of biotechnology and proteomics has become the LC / MS in particular for the separation and structure elucidation of Proteins have become one of the most important analytical techniques.

In The LC / MS of proteins and peptides is electrospray ionization (ESI) the most used Ionization technology, in which the addition of ionization aids or ionization additives proven Has. These ionization additives facilitate the transition of the analytes to charged ones Particles (ions) in the gas phase, as described in LC-GC Europe, 17 (12), Pp 646-649, 2004, is described. One of the most common additives in the normal HPLC is the phosphoric acid, and their Na or K salts, since they have a. Due to the 3 buffer stages allow excellent pH adjustment. The salts of phosphoric acid are but not fleeting, support so that the ionization does not and are deposited as precipitation on Shield the ionization chamber from where it is sometimes consuming again must be removed.

Suitable ionizing additives have been found to be organic acids such as formic, acetic and propionic acids. However, the separation then takes place in the acid and almost exclusively positive ionization to form [M + H] ⁺ ions.

When mild alternative has been the use of ammonium salts of Formic and acetic acid, especially of ammonium acetate proved to be advantageous; This can be adjusted to pH values, which is a separation and ionization under mild conditions, as well as positive and negative ionization allow. In addition, it is characterized by a good volatility from, i. it stays under the typical ESI ionization conditions of about 350 ° C At atmospheric pressure hardly or no residues in the ionization chamber back.

A Another difficulty is the presence of alkali ions already in low concentrations of 5-6 ppm the absolute sensitivity clearly impair (See also LC-GC Europe, 17 (12), pp. 646-649, 2004). These alkali ions can due to insufficiently pure starting materials or during use of water or methanol also by washing out of the glass walls in the solvent be introduced. With acetonitrile, the glass undergoes such a Washing to a much lesser extent; it is u.a. therefore preferred used.

Therefore would it be advantageous mixture of an ionizing additive and a solvent so-called solvent blend, to disposal to put that no residue in the Ionization chamber leaves and as possible is low in alkali ions.

however be salt-like ionizing additives, such as ammonium acetate containing Blends in acetonitrile previously only with the addition of 5 to 10% water produced since ammonium salts of lower organic acids, such as formic acid, acetic acid or propionic acid, not soluble in pure acetonitrile and water is needed as a co-solvent. Next it is necessary to prepare these mixtures fresh shortly before use, as below a water content of 5%, the solvent mixture containing ammonium acetate due to poor storability and pH stability because of the solution escaping ammonia. This high water content also leads Increased to the previously mentioned undesirable phenomenon washing out.

Further it is not possible with aqueous acetonitrile blends, one Gradient value of 100% acetonitrile. Such is for example with an ammonium acetate-containing blend in acetonitrile, the made with the addition of 10% water, only one gradient from 0 to 90% acetonitrile feasible.

task The invention therefore was to contain salt-like ionization additives Blends in acetonitrile available to provide, which are low in water and sodium, by a good storage stability and pH stability characterized and little or no residues in the ionization chamber leave a mass spectrometer.

These Task is solved by adding an organic acid in excess to the salt ionizing additive is added so that the base is kept in the protonated state can be. Next, the added organic acid also works as a co-solvent, allowing the ionization additive even in the absence soluble in water is.

When acid component acids suitable for the salt ionizing additive include volatile organic compounds Acids. Sufficiently volatile Acids have a boiling point at normal pressure below 300 ° C, more preferred below 250 ° C and most preferably below 200 ° C. Preferred examples are formic acid, Acetic acid, propionic acid and Trifluoroacetic acid.

Suitable bases as the base component of the salt ionization additive include volatile ones weak bases. Sufficient volatile bases have a boiling point at normal pressure below 300 ° C, more preferably below 250 ° C, and most preferably below 200 ° C. Preferred examples are ammonia, methylamine, ethylamine, n-propylamine, isopropylamine, dimethylamine, diethylamine, trimethylamine and triethylamine.

When Co-solvent organic acids include volatile organic compounds Acids. Sufficiently volatile acids have a boiling point at normal pressure below 300 ° C, more preferably below 250 ° C and most preferably below 200 ° C. Preferred examples are formic acid, acetic acid and propionic acid.

The Ionization additive can be used in any amount up to the solubility limit of the additive in the blend. It is preferable that Ionization additive in an amount of 0.001 to 3% (w / v), more preferably from 0.01 to 2% (w / v), and most preferably from 0.1 to 1% (w / v).

The Co-solvent organic acid may be present in any amount be used. It is preferred to use the organic acid in one Amount of up to 1% by volume, based on the volume of acetonitrile, more preferably 8 to 32 equivalents, based on the molar amount of the ionization additive, and most preferably 10 to 16 equivalents, to use., The water content of the LC / MS mixtures according to the invention is preferably between 0 and 5%, more preferably between 0.1% and 4%, more preferably between 0.5% and 3%, and most preferably between 1% and 2%, and may be according to the usual Methods, e.g. Karl Fischer titration.

The Purity of the substances used is preferably so high that the use of the mixtures according to the invention in the high-performance liquid chromatography (HPLC) or the LC / MS possible is. In particular, it is preferred that the substances used are so pure that organic impurities such as e.g. plasticizers, are essentially absent, i. the content of organic impurities is less than 10 ppm, more preferably less than 6 ppm and most preferably less than 1 ppm. It is also preferred that the used Substances are so pure that the content of alkali and alkaline earth ions less as 5 ppm per ionic species, more preferably less than 2 ppm per ionic species and most preferably less than 1 ppm per ion species.

The Process of preparation, in particular the mixing order, subject no restrictions. In particular, it is possible Add the ionizing additive as a salt or salt through separate addition of the acid and base during production in situ. The receiving product can optionally sterilized by filtration.

Description of the drawings

• A: Wasser; 0,1 % Ammoniumacetat/reines Acetonitril
• B: Wasser; 0,1 % Ammoniumacetat/Methanol; 0,1 % Ammoniumacetat
• C: Wasser; 0,1 % Ammoniumacetat/Acetonitril; 0,1 % Ammoniumacetat
• D: reines Wasser/Acetonitril; 0,1 % Ammoniumacetat

The 1 shows the pH gradients of mixtures according to the invention and of comparative mixtures. The pH gradients A, B, C and D consist of the following components:

• A: water; 0.1% ammonium acetate / pure acetonitrile
• B: water; 0.1% ammonium acetate / methanol; 0.1% ammonium acetate
• C: water; 0.1% ammonium acetate / acetonitrile; 0.1% ammonium acetate
• D: pure water / acetonitrile; 0.1% ammonium acetate

The 2 shows the MS total ion chromatogram of the separation of bradykinins. The peaks 1 to 5 stand for:

1

BK 1-6

2

Lys-Ala3-BK

3

BK

1

Des-Arlg-BK

5

pollution

With the LC / MS mixtures according to the invention, it is possible to realize pH gradients from pH 3.5 to pH 6.5, which permit positive and negative ionization during the chromatographic separation. As based on the 1 can be seen, a steadily increasing gradient of pH 3.5 to pH 6.5 can be realized.

The solvent mixtures according to the invention for LC / MS make possible the chromatographic separation of very similar peptides (bradykinins (BK)) under slightly acidic conditions ( 2 , upper chromatogram, conditions C). This separation is significantly worse under conventional neutral to slightly alkaline conditions ( 2 , lower chromatogram, conditions A).

at the operation of the ESI ionization chamber with the mixtures according to the invention a significantly reduced deposit of residues was found.

in the Connection are production examples of inventive LC / MS blends listed. Refer to the numbers in parentheses after the starting materials refer to the product numbers of the Fluka-Riedel catalog from the year Of 2005.

Production Example 1

One 0.1% (w / v) ammonium acetate containing ionization additive LC / MS blend was prepared as follows.

In In a glass apparatus, one part by volume of an aqueous 10% by weight ammonium acetate solution (# 32301, # 34877) and stirred one volume of acetic acid (# 33209) added. Subsequently Add 100 volumes of acetonitrile (# 34697) to a Blend for LC / MS with an ionization additive concentration of 0.1% (w / v) to obtain. The mixture is sterile filtered through a 0.2 μm filter. The has obtained clear colorless liquid a water content, determined by Karl Fischer titration, from under 2.0% and a Na content of less than 2 ppm and a K, Mg and Ca content of less than 0.5 ppm.

Production Example 2

One 0.05% (w / v) ammonium acetate containing ionization additive LC / MS blend was like follows.

In of a glass apparatus are 100 liters of acetonitrile (# 34967) and one part by volume Acetic acid (# 33209) submitted and cooled. Subsequently be stirred 0.65 mol of ammonia introduced to form a blend for LC / MS with an ionization additive concentration of 0.05% (w / v). The resulting clear colorless liquid has a water content, determined by Karl Fischer titration, of less than 0.01% and a Na content of less than 1 ppm and one K, Mg and Ca content of less than 0.5 ppm.

Claims (14)

Solution of a salt in acetonitrile, characterized in that the acid component of the salt is an organic acid boiling at less than 300 ° C at atmospheric pressure, the base component of the salt is less than 300 ° C at normal pressure boiling base, one at less than 300 ° C at normal pressure boiling organic acid in an amount up to 1 vol .-%, based on the volume of acetonitrile, is mixed and the water content, determined by Karl Fischer titration, below 5%. solution A salt according to claim 1, wherein the acid component of the salt is below formic acid, Acetic acid, propionic and trifluoroacetic acid chosen is the base component of the salt under ammonia, methylamine, Ethylamine, n-propylamine, isopropylamine, dimethylamine, diethylamine, Trimethylamine and triethylamine is chosen and those at less as 300 ° C at normal pressure boiling organic acid is selected from formic acid, acetic acid and propionic acid. solution according to one of the claims 1 or 2, wherein the boiling at less than 300 ° C at atmospheric pressure organic Acid in an amount of 8 to 32 equivalents, based on the amount of mol of the salt, is mixed. solution according to one of the claims 1 to 3, wherein the water content is less than 2. solution according to one of the claims 1 to 4, wherein the salt is ammonium formate, ammonium acetate and Ammonium propionate selected is. solution according to one of the claims 1 to 5, wherein the acetonitrile has HPLC purity or LC-MS purity. solution according to one of the claims 1 to 6, the solution was sterile filtered. solution according to one of the claims 1 to 7, wherein the proportion of alkali and alkaline earth ions below 2 ppm per inner species. solution according to one of the claims 1 to 8, the solution contains less than 10 ppm of organic contaminants. solution according to one of the claims 1 to 9, the solution containing the salt in an amount of 0.001 to 3% (w / v). solution a salt, available by the acid component a salt, chosen below at less than 300 ° C at normal pressure boiling organic acids, the base component of a Salt, chosen below at less than 300 ° C at normal pressure boiling bases, and one at less than 300 ° C at atmospheric pressure boiling organic acid, used in an amount of up to 1% by volume, based on the volume of acetonitrile, be mixed, the water content, determined by Karl Fischer Titration of the resulting mixture is less than 5%. solution according to claim 11, wherein the acid component and the base component are added together as a salt. A solution according to any one of claims 11 or 12, wherein the acid component of the salt is selected from formic acid, acetic acid, propionic acid and trifluoroacetic acid, the base component of the salt among ammonia, methylamine, ethylamine, n-propylamine, isopropylamine, dimethylamine, diethylamine, trimethylamine and triethylamine is selected and boiling at less than 300 ° C at atmospheric pressure organic acid is selected from formic acid, acetic acid and propionic acid. solution according to one of the claims 11 to 13, the solution was sterile filtered.