EP1913378A1

EP1913378A1 - Lc/ms blends containing ionising additives

Info

Publication number: EP1913378A1
Application number: EP06761828A
Authority: EP
Inventors: Joachim Emmert
Original assignee: Sigma Aldrich Production GmbH
Current assignee: Sigma Aldrich Production GmbH
Priority date: 2005-07-29
Filing date: 2006-07-27
Publication date: 2008-04-23
Also published as: WO2007012319A1; US20090306341A1; DE112006002660A5

Abstract

The invention relates to the preparation of a salt in acetonitrile, characterised in that the acid component of the salt is an organic acid which boils at less than 300 °C under normal pressure, the base component of the salt is a base which boils at less than 300 °C under normal pressure, an organic acid which boils at less than 300 °C under normal pressure is added in a quantity of up to 1 vol. %, in relation to the volume of acetonitrile, and the water content, that can be determined by Karl Fischer titration, is below 5 %.

Description

ionizing additives containing LC / MS blends

LC / MS, the coupling of liquid chromatography with mass spectrometry, has been the method of choice since the 1990s for a wide variety of issues in pharmaceutical, clinical and forensic chemistry, as well as in environmental analysis and food monitoring. Particularly in the field of biotechnology and proteomics, LC / MS has become one of the most important analysis techniques, in particular for the separation and structure elucidation of proteins.

In the LC / MS of proteins and peptides, electrospray ionization (ESI) is the most commonly used ionization technique, in which the addition of ionization aids or ionization additives has proven itself. These ionization additives facilitate the transition of the analytes to charged particles (ions) in the gas phase, as described in LC-GC Europe, 17 (12), p. 646-649., 2004. One of the most common additives in normal HPLC is the phosphoric acid and its sodium or potassium salts, since they allow an excellent pH adjustment due to the 3 buffer stages. However, the salts of phosphoric acid are not volatile, thus do not support the ionization and deposited as a precipitate on the shield of the ionization chamber, from where they need to be partially removed again consuming.

Suitable ionizing additives have been found to be organic acids such as formic, acetic and propionic acids. However, then the separation takes place in the acid and almost exclusively positive ionization to form [M + H] ⁺ - ions. As a mild alternative, the use of the ammonium salts of formic and acetic acid, in particular of ammonium acetate has been found to be advantageous; This allows adjustment of pH values which allow separation and ionization under mild conditions, as well as positive and negative ionization. In addition, it is characterized by a good volatility, ie remain under the typical ESI ionization conditions of about 35O ⁰ C at atmospheric pressure hardly or no residues in the ionization chamber back.

Another difficulty is the presence of alkali ions, which even at low concentrations of 5-6 ppm, the absolute sensitivity significantly affect (see also LC-GC Europe, 17 (12), p 646-649, 2004). These alkali ions can be introduced by not sufficiently pure starting materials or by the use of water or methanol by washing out of the glass walls in the solvent. With acetonitrile, the glass undergoes such leaching to a significantly lesser extent; it is u.a. therefore preferably used.

It would therefore be advantageous to provide a mixture of an ionization additive and a solvent, a so-called solvent blend, which leaves no residues in the ionization chamber and is as low in alkali ions as possible.

However, salt-like ionizing additives such as ammonium acetate-containing blends in acetonitrile have heretofore been prepared only with the addition of 5 to 10% water because ammonium salts of lower organic acids such as formic acid, acetic acid or propionic acid are not soluble in pure acetonitrile and water is co-solvent is needed. Furthermore, it is necessary to prepare these mixtures freshly shortly before use, since, below a water content of 5%, the solvent mixture comprising ammonium acetate is characterized by poor storage stability and pH stability due to escape from the solution. distinguishes ammonia. In addition, this high proportion of water increasingly results in the aforementioned undesired leaching phenomenon.

Furthermore, it is not possible with hydrous acetonitrile blends to set a gradient value of 100% acetonitrile. Thus, for example, with a blend containing ammonium acetate in acetonitrile, which was prepared with the addition of 10% water, only a gradient value of 0 to 90% acetonitrile can be realized.

The object of the invention was therefore to provide salt-containing ionization containing blends in acetonitrile available that are low in water and sodium, are characterized by good storage stability and pH stability and leave little or no residue in the ionization chamber of a mass spectrometer.

This object is achieved by adding an organic acid in excess to the salt-like ionizing additive, so that the base can be kept in the protonated state. Furthermore, the added organic acid also acts as a co-solvent so that the ionization additive is also soluble in the absence of water.

Acids useful as the acid component of the salt ionizing additive include volatile organic acids. Sufficient volatile acids have a boiling point at normal pressure below 300 ° C, more preferably below of 25O ⁰ C and most preferably below 200 ⁰ C, to. Preferred examples are formic acid, acetic acid and propionic acid.

Suitable bases as the base component of the salt ionization additive include volatile weak bases. Sufficiently volatile bases have a boiling point at normal pressure below 300 ⁰ C, more preferably below of 25O ⁰ C and most preferably below 200 ⁰ C, to. Preferred examples - A -

Ie are ammonia, methylamine, ethylamine, n-propylamine, isopropylamine, dimethylamine, diethylamine, trimethylamine and triethylamine.

Co-solvent organic acids include volatile organic acids. Sufficient volatile acids have a boiling point at normal pressure below 300 ° C, more preferably below 250 ° C and most preferably below 200 ⁰ C, to. Preferred examples are formic acid, acetic acid and propionic acid.

The ionization additive may be used in any amount up to the limit of solubility of the additive in the blend. It is preferred that the ionizing additive be present in an amount of from 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) , to use.

The co-solvent organic acid can be used in any amount. It is preferred to include the organic acid in an 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%, even more preferably between 0.5% and 3%, and most preferably between 1% and 2%. , and can according to the usual methods, eg Karl Fischer titration.

The purity of the substances used is preferably so high that the use of the mixtures according to the invention in chromatography (HPLC) and LC / MS is possible. In particular, it is preferred that the substances used are so pure that organic impurities, such as plasticizers, are substantially absent, ie 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 substances used be so pure that the content of alkali and alkaline earth ions is less than 5 ppm per ionic species, more preferably less than 2 ppm per ionic species, even more preferably less than 1 ppm per ionic species , and most preferably less than 0.2 ppm per ion species. Further, it is preferred that the total alkali content be below 0.25 ppm, more preferably below 0.2 ppm, and most preferably below 0.15 ppm.

The method of preparation, in particular the mixing order, is not limited. In particular, it is possible to add the ionization additive as a salt or to generate the salt in situ by separate addition of the acid and base during production. The resulting product can optionally be sterilized by filtration.

Description of the drawings

FIG. 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

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

1: BK 1-6 2: Lys-Ala3-BK

3: BK

4: Des-Argl-BK

5: contamination

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 can be seen from Fig.l, 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 (FIG. 2, upper chromatogram, conditions C). This separation is much worse under conventional neutral to slightly alkaline conditions (Figure 2, lower chromatogram, Conditions A).

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

Following are examples of preparation of inventive LC / MS blends listed. The numbers in parentheses after the starting materials refer to the product numbers of the 2005 Fluka Riedel catalog.

Production Example 1

A 0.1% (w / v) ammonium acetate ionizing additive-containing LC / MS blend was prepared as follows.

In a glass apparatus, a volume of an aqueous 10 wt .-% ammonium acetate solution (# 32301, # 34877) is introduced and one part by volume with stirring Acetic acid (# 33209) added. Subsequently, 98 volumes of acetonitrile (# 34697) are added to give a blend for LC / MS with an ionization additive concentration of 0.1% (w / v). The mixture is sterile filtered through a 0.2 μm filter. The resulting clear colorless liquid has a water content, determined by Karl Fischer titration, of less than 2.0% and a Na content of less than 2 ppm and a K, Mg and Ca content of less than 0.5 ppm on.

Production Example 2

A 0.05% (w / v) ammonium acetate ionizing additive-containing LC / MS blend was prepared as follows.

100 l of acetonitrile (# 34967) and one part of acetic acid (# 33209) are placed in a glass apparatus and cooled. Then, with stirring, 0.65 mol of ammonia are introduced to obtain 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 a K, Mg and Ca content of less than 0.5 ppm on.

Production Examples 3 to 8

0.1% (w / v) ammonium acetate containing ionization additive LC / MS blends were prepared as in Example 1 except that the compositions were changed as shown in Table 1 and optionally water was added. Table 1

The solutions obtained were analyzed for their alkali content by ICP-OES and ICP-MS. The results shown in Table 2 were obtained:

Table 2

It can be seen from Table 2 that the total alkali content of the inventive examples is below 0.25 ppm and preferably below 0.2 ppm. The solutions obtained according to Examples 3 to 8 were further used for the chromatographic separation of very similar peptides (bradykinins (BK)) as previously shown. The following results were obtained.

From Table 3 it can be seen that the HPLC separation of the very similar peptides is significantly better at low water and alkali ion contents.

Claims

claims

A 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 normal pressure, the base component of the salt being less than

300 ° C at normal pressure boiling base is, at less than 300 ° C at atmospheric pressure boiling organic acid in an amount up to 1 vol .-%, based on the volume of acetonitrile, mixed and the water content, determined by Karl Fischer Titration is less than 5%.

2. Solution of a salt according to claim 1, 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 under ammonia, methylamine, ethylamine, n-propylamine, isopropylamine, dimethylamine, diethylamine, trimethylamine and Triethylamine is selected and at less than 300 ⁰ C at

Normal pressure boiling organic acid is selected from formic acid, acetic acid and propionic acid.

3. A solution according to any one of claims 1 or 2, wherein the at less than 300 ° C at atmospheric pressure boiling organic acid in an amount of 8 to

32 equivalents, based on the molar amount of the salt, is mixed.

4. A solution according to any one of claims 1 to 3, wherein the water content is less than 2%.

5. A solution according to any one of claims 1 to 4, wherein the salt is selected from ammonium formate, ammonium acetate and ammonium propionate.

A solution according to any one of claims 1 to 5, wherein the acetonitrile has HPLC purity or LC-MS purity.

7. A solution according to any one of claims 1 to 6, wherein the solution has been sterile filtered.

8. A solution according to any one of claims 1 to 7, wherein the proportion of alkali and alkaline earth ions is less than 2 ppm per ionic species.

A solution according to any one of claims 1 to 8, wherein the solution contains less than 10 ppm of organic impurities.

A solution according to any one of claims 1 to 9, wherein the solution contains the salt in an amount of 0.001 to 3% (w / v).

11. A solution of a salt obtainable by adding the acid component of a salt selected from organic acids boiling at less than 300 ° C at normal pressure, the base component of a salt chosen among less than

300 ° C at atmospheric pressure boiling bases, and at less than 300 ° C at normal pressure boiling organic acid, used in an amount up to 1 vol .-%, based on the volume of acetonitrile, mixed, the water content, determined by Karl Fischer titration, the resulting mixture is less than 5%.

The solution of claim 11, wherein the acid component and the base component are added together as a salt.

13. The solution according to claim 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 is ammonia, methylamine, ethylamine, n-propylamine, isopropylamine, dimethylamine, diethylamine. min, trimethylamine and triethylamine is selected and which is chosen at less than 300 ° C at normal pressure boiling organic acid with formic acid, acetic acid and propionic acid.

14. A solution according to any one of claims 11 to 13, wherein the solution has been sterile filtered.

15. A method for chromatographic separation, characterized by the use of the solution according to any one of claims 1 to 14.

16. A method for chromatographic separation according to claim 15, wherein peptides are chromatographed.

17. Use of the solution according to one of claims 1 to 14 in chromatography.

18. Use according to claim 17, wherein peptides are chromatographed.