WO2014098526A1 - Quantification method for sulphur-containing organic compound - Google Patents

Abstract

The present invention relates to a quantification method for a sulphur-containing organic compound. Sulphur containing compounds can be quantified by means of the quantification method of the present invention without limitation, but, preferably, the present invention is relatively effective in quantifying organic compounds that are difficult to quantify such as those which are hygroscopic or take the form of hydrides, or else macromolecules such as biological materials. Further, among biological materials, the quantification method of the present invention can highly effectively quantify peptides or peptide high-purity standard substances.

Description

Determination of Sulfur-Containing Organic Compounds

The present invention relates to a method for quantifying sulfur-containing organic compounds.

The present invention relates to a method for quantifying sulfur-containing organic compounds. In general, the accuracy and traceability of quantitative analysis is very important as a measure of the reliability of experimental results and the possibility of mutual comparison between experiments. In particular, the reliability of the measurement can be seriously affected when the acquisition of high purity standards with traceability in the quantitative analysis of organic compounds and biomaterials is not preceded. Quantitative values of high-purity standards or solutions are prepared by gravimetric methods and based on corrected purity, based on the results of various purity analyses for the material. Alternatively, in the case of complex substances such as biomolecules, high purity standards of organic compounds such as nucleic acid monomers / amino acids, which are the basic units of biomolecules, are secured in the same manner as above and used as a reference to characterize high purity biomaterials The method of obtaining a quantitative value is used. For example, in the case of protein standard / standard solution quantification, a method of quantitating peptides by enzymatic digesting of proteins in peptide units is also widely used, but for this, a peptide standard having an accurate content is required. In the case of peptide or protein high purity standards, the method of determining the content of peptide or protein by quantifying the amino acid generated by hydrolysis of each is widely used ( J. Chromatography A , 1218 , 6596-6602 (2011)). However, in order to use this method, a standard of amino acid used for quantification must be secured and hydrolysis conditions must be established to ensure that the efficiency of the protein / peptide hydrolysis to amino acids is close to 100%. As an example of a technique for establishing a method for quantifying the biological material, in order to quantify DNA, an attempt has been made to cut an nucleic acid monomer using an enzyme and quantify them by isotope dilution HPLC-MS. (O'Connor, G., et. Al., Anal. Chem. , 74 , 3670-3676 (2002)) Also, for proteins, UV absorbance measurement method, Biuret method, BCA method, Lowry method or Bradford method Protein is quantified mainly using the back light, but the situation of sensitivity and dynamic range (dynamic range) has not been solved. In addition, since the mass of the organic compound having high hygroscopicity or a hydrate form varies depending on the number of water molecules included in the sample, it is very difficult to accurately measure the mass.

It is an object of the present invention to provide a method for quantifying sulfur containing organic compounds. More specifically, the present invention provides a method for quantifying a biological material selected from a sulfur-containing organic compound, methionine, cysteine or both, or a peptide or protein including sulfur and DNA, RNA or PNA modified to include sulfur. In particular, when the organic compound or the biomaterial is present in the form of a hydrate or hygroscopic, it is effective to solve the problem that the reliability of the manufactured value manufactured by weight method from the high purity organic compound or standard material whose purity is confirmed by the conventional method is effective. Provide means.

The present invention relates to a method for quantifying sulfur-containing organic compounds using a sulfur isotope ratio. More specifically, the present invention relates to a method for quantifying sulfur-containing organic compounds including the following steps (1) to (4).

(1) A sample solution containing sulfur-containing organic compounds is diluted with an internal standard solution in which any one isotope selected from ³³ S, ³⁴ S and ³⁶ S is concentrated. Preparing a sample mixture solution such that a theoretical isotope ratio with ³² S) is between 0.2 and 5;

(2) A mixed solution prepared in step (1) by mixing a theoretical standard isotope ratio by mixing the internal standard solution having any one isotope selected from ³³ S, ³⁴ S and ³⁶ S with a sulfur standard solution having a natural presence ratio Preparing a correction solution to be equal to the value;

(3) recovering the sulfur oxide (sulfate) in inorganic form by decomposing the organic compound containing sulfur in the mixed solution prepared in step (1); And

(4) measuring the isotope ratio of any one isotope and ³² S selected from ³³ S, ³⁴ S and ³⁶ S of the sulfur oxide recovered in step (3), Measuring the corresponding isotope ratio of the calibration solution prepared in step (2).

The sulfur-containing organic compound is not only an organic compound such as methionine, cysteine, but also a protein or peptide including methionine, cysteine or both.

In the above step (3), it is preferable to decompose the sulfur oxide in the form of an inorganic substance by simultaneously treating the sample mixture solution containing sulfur-containing organic compounds with electromagnetic radiation and acid decomposition. Treatment of the electromagnetic wave irradiation and acid decomposition at the same time includes the acid decomposition while maintaining at 100 to 300 ℃ for 10 minutes to 240 minutes in an electromagnetic oven, and further repeat the electromagnetic oven treatment. Simultaneous treatment of microwave irradiation and acid decomposition can effectively decompose sulfur into inorganic elements, but not limited thereto, and sulfur may be decomposed into inorganic materials under various conditions. In order to decompose the acid, it is necessary to add enough oxidizing agent to convert all the elements of the organic compound into the inorganic element form. When the total moles of carbon, hydrogen, nitrogen, and sulfur atoms constituting the organic compound containing sulfur are based on 1, it is preferable to use the molar ratio of the oxidizing agent at least 10 times or more. The oxidizing agent is characterized by using an oxidizing agent other than sulfuric acid, a preferred example is nitric acid, perchloric acid, hydrogen peroxide or a mixture thereof.

If an oxidizing agent of less than 10 times molar ratio is added, the acid decomposition may not occur sufficiently, and if exceeded, there is no big problem, but the acid concentration of the final assay solution does not exceed 10% by weight in the ICP / MS analysis. Suitable.

Isotope ratio of any one isotope and ³² S selected from ³³ S, ³⁴ S and ³⁶ S of the sample mixture solution obtained in step (4) and It is characterized by obtaining the concentration of the sample solution to be measured by applying the corresponding isotope ratio of the correction solution to <Equation 1>.

<Equation 1>

C _x : concentration of the sample solution (x) to be measured,

m _x : mass of the sample solution (x) measured using a balance,

m _y : Mass of the internal standard solution (y) in which any one isotope selected from ³³ S, ³⁴ S and ³⁶ S added to the sample solution (x) is concentrated

m _y ': Mass of the internal standard solution (y) in which any one isotope selected from ³³ S, ³⁴ S and ³⁶ S is added to the calibration sulfur standard solution (z)

C _z : Concentration of calibration sulfur standard solution (z)

R _x : Sulfur isotope ratio present in sample solution (x) (any isotope selected from ³³ S, ³⁴ S and ³⁶ S / ³² S isotope)

R _y : Sulfur isotope ratio present in ³⁴ S concentrated internal standard solution (y) (any isotope selected from ³³ S, ³⁴ S and ³⁶ S / ³² S isotope)

R _z : Isotope ratio of sulfur in sulfur standard solution (z) (any isotope selected from ³⁴ S, ³³ S and ³⁶ S / ³² S isotope)

R _xi : I-isotope ratio of sulfur in sample solution (x) ( ³² S / ³² S, ³³ S / ³² S, ³⁴ S / ³² S, ³⁶ S / ³² S)

R _zi : i-isotopic ratio of sulfur present in the calibration sulfur standard solution (z) ( ³² S / ³² S, ³³ S / ³² S, ³⁴ S / ³² S or ³⁶ S / ³² S)

R _b : Sulfur isotope ratio of the sample mixture solution (any one isotope selected from ³⁴ S, ³³ S and ³⁶ S / ³² S isotope)

R _{b '} : Sulfur isotope ratio of the correction solution (any one isotopically selected from ³³ S, ³⁴ S and ³⁶ S / ³² S isotope)

w: dry mass correction factor

C _blank : concentration of _blank sample

The present invention relates to a method for quantifying sulfur-containing organic compounds. In more detail, a sample mixture solution is prepared by adding a sulfur solution in the form of an inorganic substance in which one of sulfur isotopes (of ³³ S, ³⁴ S and ³⁶ S) is concentrated to the organic compound to be quantified as an internal standard. In addition, the present invention relates to a method for quantifying organic compounds by preparing a calibration solution containing the same internal standard and then comparing and measuring the isotope ratios of the sample mixture solution and the calibration solution. The present invention can be very useful for the determination of macromolecules, particularly peptides or proteins containing methionine or cysteine, and also very useful for the determination of hygroscopic or hydrate-type organic compounds that are difficult to accurately quantify. Quantitative method.

The quantitative method of the present invention can provide highly reliable and consistent quantitative results for high-purity primary standards such as organic compounds or proteins / peptides, and thus can be utilized for characterization and establishment of measurement standards for high-purity standard solutions. The standard solution thus prepared can be used as a standard solution for calibration for quantitative analysis of various organic compounds and biomolecules, and can be utilized to secure reliability of quantitative results.

Figure 1 (A) is a schematic diagram showing a sample mixture solution (b) consisting of a sample solution (x) having a natural abundance ratio of sulfur ( ³⁴ S / ³² S) and a ³⁴ S high concentration internal standard solution (y), ( b) is a schematic view showing a calibration solution (b ') consisting of a sulfur element standard solution (z) and S ³⁴ highly concentrated internal standard solution (y).

2 is a conceptual diagram of a method for quantifying sulfur-containing organic compounds using isotope dilution inductively coupled plasma mass spectrometry (ICP / MS).

Figure 3 is a graph showing the relationship between the isotope ratio and the error magnification factor (EMF) of the sample mixture solution and the correction solution when the sulfur isotope dilution. R is the sulfur isotope ratio ( ³⁴ S / ³² S).

Figure 4 shows the quantitative results of the phosphorus growth hormone based sulfur isotope measurement.

(A) is the quantification result of phosphorus growth hormone prepared in the first batch, (B) is a comparison of sulfur isotope based quantification and amino acid based quantification results, I, F, P, V is the protein level at the amino acid level, respectively After hydrolysis, isoleucine, phenylalanine, proline and valine were quantified to calculate the protein content.

5 is a SEC-UV chromatogram of phosphorus growth hormone. The main peak of 3.3 minutes is the peak of phosphorus growth hormone and the small peak of 4.1 minutes corresponds to the small molecule.

FIG. 6 shows the results of monitoring only m / z 32 and 34 corresponding to elemental sulfur by connecting SEC separated components to ICP / MS for the analysis of the small amount of sulfur-containing impurities contained in the solvent. There is no peak due to small sulfur-containing impurities after the peak corresponding to the 3.3 minute phosphorus growth hormone molecule.

7 is a sulfur isotope-based quantitative result and amino acid-based quantitative result of the phosphorus growth hormone prepared in the second batch.

8 is a sulfur isotope-based quantification results and amino acid-based quantification results of the phosphorus growth hormone T2 peptide.

9 is a sulfur isotope-based quantitative result and amino acid-based quantitative result of the phosphorus growth hormone T11 peptide.

10 is a sulfur isotope based quantification result of methionine.

11 is a sulfur isotope based quantification result of the NIST SRM 2389a sample according to the sample pretreatment method.

12 is a result of homogeneity evaluation of phosphorus growth hormone prepared in the secondary batch.

Figure 13 is a sulfur isotope-based authentication result and amino acid or peptide-based quantification results of the phosphorus growth hormone prepared in the second batch.

The present invention relates to a method for quantifying sulfur-containing organic compounds using a sulfur isotope ratio. More specifically, the present invention relates to a quantitative method of measuring a sulfur isotope ratio contained in an organic compound and calculating an amount of sulfur-containing organic compound from the measured sulfur isotope ratio. The method for measuring the sulfur isotope ratio contained in the organic compound is preferably, but not limited to, using an inductively coupled plasma mass spectrometer.

There are four known isotopes of sulfur in nature: ³² S, ³³ S, ³⁴ S, and ³⁶ S. Among them, ³² S, the most isotope, is 95.02% and ³⁴ S is 4.21%. The present invention is characterized by using the ratio of any one isotope selected from the sulfur isotopes ³³ S, ³⁴ S and ³⁶ S and ³² S contained in the organic compound in the sample solution (x), and more specifically, the sample solution. to ³³ S, ³⁴ S and ³⁶ S any one of the isotopes is a into the concentrated internal standard solution (y) was diluted sample mixed solution (b) of ³³ S, ³⁴ S and ³⁶ S any one of isotopes selected from the group consisting of selected from the group consisting of to use an element / ³² S isotope ratio (R _b) and a sulfur standard solution (z) with the same correction consisting of an internal standard solution (y) solution (b ') the isotopic ratio (R _b') of the .

A preferred embodiment of the method for quantifying sulfur-containing organic compounds using the sulfur isotope ratio of the present invention (any one isotope selected from ³³ S, ³⁴ S and ³⁶ S / ³² S isotope) is

(1) a sample solution (x) comprising a containing organic compound of sulfur ³³ S, ³⁴ S and ³⁶ S is diluted with any one of the internal standard solution, the isotope concentration (y) selected from the theoretical isotope ratio ⁽³³ Preparing a sample mixture solution (b) such that any one isotope selected from S, ³⁴ S and ³⁶ S / ³² S isotope is 1;

(2) Simultaneously with step (1), sulfur standard solution (z) having a sulfur isotope ratio in nature and internal standard solution (y) in which any one isotope selected from ³³ S, ³⁴ S and ³⁶ S is concentrated Preparing a correction solution (b ') by mixing the theoretical isotope ratios (any one isotope selected from ³³ S, ³⁴ S, and ³⁶ S / ³² S isotope) to a value of 0.2 to 5;

(3) decomposing and recovering sulfur in the sulfur-containing organic compound of (1) to sulfur oxide (sulfate) in inorganic form; And

(4) any one isotope / ³² S isotope ratio selected from ³³ S, ³⁴ S and ³⁶ S of the sample mixture solution recovered in step (3); Measuring the isotope ratio of the calibration solution prepared in step (2).

In the quantitative method of the present invention, sulfur-containing organic compounds can be quantified without limitation, but hygroscopic or hydrate forms that have a high possibility of deflection in quantification using conventional chemical balances, mass spectrometers, or other analytical equipment It is preferable to quantify organic compounds or biomaterials. The preferred biomaterials include, but are not limited to, DNA, RNA or PNA modified to include peptides, proteins or sulfur. The peptide and protein are peptides or proteins comprising methionine, cysteine or both. The protein consists mainly of carbon, hydrogen and oxygen, and contains a low percentage of sulfur. In addition, it may contain a heteroatom (heteroatom), such as phosphorus, but it appears only in a modified form of a specific protein. Although carbon, hydrogen, and oxygen are elements having a high relative abundance in the composition of protein, it is often present in air or a solvent used for measurement, and thus it may be difficult to control the background signal. Therefore, absolute quantitation of proteins based on sulfur measurement, which is small in abundance but is present in the form of cysteine or methionine in most proteins, is desirable.

In the step (3), a method of decomposing sulfur in an organic compound containing sulfur into sulfur oxide (sulfate), which is an inorganic substance, is performed by simultaneously treating the sample mixture solution (b) with electromagnetic wave irradiation and acid decomposition. The sulfur in the compound is decomposed into sulfur oxide (sulfate) in an elemental form. More specifically, the acid-treated sample mixture solution (b) is placed in an electromagnetic oven and maintained at 100 to 300 ° C. for 10 to 240 minutes. Decomposing and further repeating the electromagnetic oven treatment. Simultaneous treatment of electromagnetic radiation and acid decomposition can be used to effectively decompose sulfur into inorganic elements, but not limited thereto, and sulfur may be decomposed into inorganic materials under various conditions. In addition, the acid that can be used in the acid decomposition is preferably nitric acid except per sulfuric acid, perchloric acid, but not limited thereto, and may be subjected to acid decomposition under conditions further including hydrogen peroxide (H ₂ O ₂ ) to increase the oxidizing power.

Based on the total moles of carbon, hydrogen, nitrogen and sulfur atoms of the organic compound to be decomposed to 1, it is preferable to use the molar ratio of the oxidizing agent at least 10 times or more. If an oxidizing agent of less than 10 molar ratio is added, the acid decomposition may not occur sufficiently, and if it exceeds, there is no big problem, but it is recommended that the acid concentration of the final assay solution is not more than 10% suitable for ICP / MS analysis. good.

Isotope ratio / ³² S isotope ratio selected from ³³ S, ³⁴ S and ³⁶ S of the sample mixture solution obtained in step (4) Once the corresponding isotope ratio of the calibration solution is obtained, the protein content in terms of sulfur content can be calculated by the following <Equation 1>.

In Equation 1, R _x and R _z may be directly measured using a natural existence ratio of 0.042 / 0.9501 ( ³⁴ S / ³² S) given sulfur in IUPAC or using a mass spectrometer for isotope ratio measurement.

<Equation 1>

C _x : concentration of the sample solution (x) to be measured,

m _x : mass of sample or sample solution (x)

m _y : Mass of the internal standard solution (y) in which any one isotope selected from ³³ S, ³⁴ S and ³⁶ S added to the sample solution (x) is concentrated

m _y ': Mass of the internal standard solution (y) in which any one isotope selected from ³³ S, ³⁴ S and ³⁶ S is added to the calibration sulfur standard solution (z)

C _z : Concentration of calibration sulfur standard solution (z)

R _x : Sulfur isotope ratio present in sample solution (x) (any one isotope selected from ³³ S, ³⁴ S and ³⁶ S / ³² S isotope)

R _y : Sulfur isotope ratio present in ³⁴ S concentrated internal standard solution (y) (any isotope selected from ³³ S, ³⁴ S and ³⁶ S / ³² S isotope)

R _z : Isotope ratio of sulfur in sulfur standard solution (z) (any isotope selected from ³⁴ S, ³³ S and ³⁶ S / ³² S isotope)

R _xi : i-isotopic ratio of sulfur present in the sample or sample solution (x) ( ³² S / ³² S, ³³ S / ³² S, ³⁴ S / ³² S, ³⁶ S / ³² S)

R _zi : i-isotopic ratio of sulfur present in the calibration sulfur standard solution (z) ( ³² S / ³² S, ³³ S / ³² S, ³⁴ S / ³² S or ³⁶ S / ³² S)

R _b : Sulfur isotope ratio of the sample mixture solution (any one isotope selected from ³⁴ S, ³³ S and ³⁶ S / ³² S isotope)

R _{b '} : Sulfur isotope ratio of the correction solution (any one isotopically selected from ³³ S, ³⁴ S and ³⁶ S / ³² S isotope)

w: dry mass correction factor

C _blank : concentration of _blank sample

Sulfur in the form of impurities may be present in reagents, solvents and containers added during sample pretreatment. Prepare a _blank sample to obtain a C _blank (sulfur concentration of the base sample) and subtract C _blank from the sulfur concentration measured in the sample. The concentration of sulfur in pure samples can be determined.

The solvent used in the present invention is preferably deionized water, a dilute acid solution or buffer solution made of deionized water, and the amount of sulfur contained in them is preferably 0.02 mg / kg or less, more preferably 10 ng / kg or less. It contains sulfur. The sulfur content in the sample determined as described above may be used to calculate the content of the organic compound using the molecular formula of the organic compound. However, since the sulfur content in the sample obtained as described above may include other impurities containing sulfur in addition to the analyte, sulfur-containing impurities should be quantified and subtracted from the total sulfur content to determine traceability in SI units. Can be equipped. The method for measuring sulfur-containing impurities is preferably, but not limited to, CE-ICP / MS or SEC-ICP / MS for proteins. In the following examples, impurities were quantified by UV and ICP / MS using a BioSep SEC-3000 column (300 x 4.6 mm) and 50 mM ammonium bicarbonate as an eluent at a flow rate of 1 ml / min.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples. These examples are only for illustrating the present invention in more detail, it is obvious to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1: Quantitative (primary) determination of phosphorus growth hormone protein based on elemental sulfur.

Sulfur content in organic compounds was completely decomposed into inorganic form to measure sulfur content, sulfur-containing impurities were evaluated through SEC-ICP / MS, and sulfur content measurement based organic compound quantification method with SI traceability was performed. Phosphorus growth hormone has three methionine and four cysteines with an average molecular weight of 22,125 Da and contains seven sulfur atoms per molecule. Thus, one mole of hGH corresponds to seven moles of sulfur.

(1) Preparation of Mixed Solution and Calibration Solution (See FIG. 1 and FIG. 2)

 ① Phosphorus Growth Hormone (hGH) standard solution (concentration of about 65 mg / kg in sulfur content in the first batch) of 0.5 g from each of five vessels (vial) were collected in each of the five Teflon-based vessels (TFM vessel).

② Add 1 mL of 32.5 mg / kg ³⁴ S concentrated standard solution (Oak Ridge National Lab.) To the sample, and the expected isotope ratio ( ³⁴ S / ³² S) is 1 (5 sample blends) were prepared.

③ Mix 1 mL of 32.5 mg / kg sulfur standard solution with 1 mL of the above ³⁴ S concentrated standard solution, dilute to 30 g with 10% (dilution ratio) HNO ₃ , and give 5 calibration blends of isotope ratio 1. Was prepared.

 ④ 3 mL nitric acid, 3 mL deionized water, and 2 mL hydrogen peroxide water were added to a Teflon-based vessel containing a sample blend.

 ⑤ The Teflon-based vessel was sealed and placed in an electromagnetic oven, heated to 200 ° C. for 15 minutes, and maintained at 200 ° C. for 20 minutes to perform electromagnetic-assisted acid decomposition.

 ⑥ After the Teflon vessel was cooled to some extent, the vessel was shaken sufficiently to homogenize the solution inside the vessel.

 ⑦ Disassemble the TFM pressure vessel to recover the sample blend solution and dilute to 30 g with deionized water.

On the other hand, optimal isotope ratio is the goal of the so closer to 1 the isotope ratio ⁽³⁴ S / ³² S) measured deflection is minimized (see Fig. 3), the isotope ratio ⁽³⁴ S / ³² S) 1 isotope dilution Isotope ratio ( ³⁴ S / ³² S). Therefore, the first embodiment in the US National Institute of Standards and Technology as described above (National Institute of Standards and Tchnology; NIST) prepared by diluting the sulfur standard solution (SRM 3154) (z), available working solution and in the ³⁴ S concentration internal standard The solution (y) was mixed to prepare a calibration solution (b ') such that the isotope ratio ( ³⁴ S / ³² S) was 1 (see FIGS. 1 and 2).

(2) ICP / MS measurement

① Thermo's high resolution ICP / MS was optimized for high resolution (R> 10,000). (Medium resolution is sufficient to eliminate ionic interference from acids, solvents, organics and ICP gases, but the device sensitivity is improved with high resolution to minimize the effects of high background signals of deionized water and sulfur in the used acid and apparatus. Lower measurement): The prepared sample blend (b) and calibration blend (b ') showed signal strengths of about 600,000-700,000 cps for ³² S and ³⁴ S.

② Isotopically measured isotopes while sweeping ³² S and ³⁴ S at high speeds, and isotopic ratios of known isotope ratio standard samples were periodically measured together with ID-ICP / MS. The bias correction and drift were corrected by bracketing correction.

③ The measurement uncertainty was calculated using the measured isotope ratio, and the uncertainty of the estimation of each factor affecting the measurement was synthesized to estimate the measurement uncertainty.

(3) Result of measurement of hGH primary batch sulfur content by ID-ICP / MS

The hGH primary batch had a manufacturer-supplied concentration of 303 μmol / kg and was determined to be 290 μmol / kg (65 mg / kg sulfur content) based on amino acid based quantification.

  ① The sulfur content measurement results of the hGH primary batch are shown in Table 1 and FIG. 4A. Compared to the amino acid based quantitative result of the conventional method, the sulfur based quantitative result was about 5% higher.

Table 1. Sulfur measurement results of the first batch of phosphorus growth hormone standard solution.

 ② In order to confirm why the sulfur element-based protein quantitative result of the present invention has a higher quantitative value than the conventional amino acid-based result, first, it is possible that small molecules including elemental sulfur in the sample existed and contributed to the total sulfur content. Size Exclusion Chromatography (SEC) -UV and SEC-ICP / MS confirmed (see Figures 5 and 6).

The small peak eluting after 4 minutes in SEC-UV is the small molecule peak, and the ion chromatogram is obtained by fixing MS to m / z 32 and 34 corresponding to sulfur ions in SEC-ICP / MS. It was confirmed that there was no detectable peak at this retention time. The results showed that the effect of sulfur-comprising small molecular impurities on the determination of total sulfur content was less than 0.5%. Therefore, it was determined that the element-based protein quantitative results were not likely to be overestimated by the sulfur-containing impurities in Example 1 of the present invention.

Example 2: Quantitative (secondary batch) determination of phosphorus growth hormone protein based on elemental sulfur.

Phosphorus growth hormone protein was quantified for the newly prepared hGH secondary batch in the same manner as in Example 1. Since the concentration of the sample was prepared about one-third lower than that of the first batch, the analytical sample volume was increased from 0.5 g to 1.0 g and the concentration of the concentrated isotope solution was adjusted accordingly. We made it as close to 1 as before. One aliquot was taken from two or more vials because the amount of sample per vial of the second prepared hGH was less than the sample volume. The analysis results of the secondary batch are shown in FIG. 7 and Table 2.

Table 2. Sulfur measurement results of the second batch of phosphorus growth hormone standard solution.

Example 3 Element-Based Absolute Quantitation of hGH T2, T11 Peptides

As the same measurement bias was observed across the secondary in the determination of elemental hGH based on sulfur, T2 of hGH, which seems to be easier to hydrolyze, to examine the possibility that amino acid-based quantitative results were affected by the efficiency and side reactions of hydrolysis. A T11 peptide standard solution was prepared to compare the results of amino acid based quantification and elemental sulfur based quantification.

T2 peptide, the second tryptic peptide from the N terminus of the phosphorus growth hormone, has the sequence of LFDNA M LR, so that there is one methionine including sulfur, so it has one sulfur atom per peptide, and thus sulfur element-based quantification is possible.

Since the T11 peptide has the same sequence as DLEEGIQTL M GR and also contains one methionine, one equivalent of the peptide contains one equivalent of sulfur atom. The concentration of T2 was prepared at 1 mmol / kg and the purity provided by the manufacturer was 99.1%, the concentration of T11 was made at 1 mmol / kg and the purity provided by the manufacturer was 99%. Thus, the expected content of elemental sulfur is 32 mg / kg.

The elemental sulfur based peptide quantification as described in FIG. 8 and Table 3 can determine the T2 peptide content to a level of 3.1% expansion uncertainty. However, the elemental sulfur based quantification results for the T2 peptide show a 10% measurement bias as compared to amino acid based quantification as well as protein quantification. The T11 peptide could be quantified with an expansion uncertainty of 0.84% but showed a similar measurement bias as the T2 peptide (see FIG. 9 and Table 3). Thus, even in the hydrolysis of peptides for amino acid based quantification, there is still no possibility that the impairment of hydrolysis and the loss due to secondary reaction of acid degradation still exist or that the observed measurement bias is due to other factors.

Table 3. Sulfur measurement results of phosphorus growth hormone T2 peptide standard solution.

Table 4. Sulfur measurement results of phosphorus growth hormone T11 peptide standard solution.

Example 4 Element-Based Absolute Quantitation of Methionine

Sulfur element-based quantification of small peptide standard solutions compared to proteins resulted in the same measurement bias as protein standard solutions, and amino acid standard solutions (Met and Cys) containing sulfur were prepared to replace purity analysis with sulfur element-based quantification and amino acids. The standard solution was certified and utilized. Thus prepared and certified amino acid standard solution can be utilized to quantify proteins and peptides on an amino acid basis using LC-MS / MS. Since cysteine can be easily changed into disulfide form by oxidation reaction, first, standard solution of methionine was prepared and elemental absolute absolute was tried to avoid the complexity of analysis. Since methionine is easily oxidized based on amino acid-based quantification, it is necessary to analyze oxidized methionine as well as primitive methionine in LC-MS / MS analysis.

The concentration of the prepared methionine standard solution was 1 mmol / kg and the purity provided by the manufacturer was 99.5% or more. This corresponds to a sulfur content of 32 mg / kg. Sulfur element-based quantitative results of the methionine standard solution are shown in FIG. 10 and Table 5, and quantitative results within 1% of the expansion uncertainty were obtained. The methionine standard solution thus prepared can be used for future amino acid-based quantification of hGH as well as proteins including T2, T11 peptide and methionine of hGH.

Table 5. Elemental sulfur based quantitative analysis of methionine standard solution.

Example 5 Validation of Existing Sample Pretreatment Methods (Acid Degradation by Electromagnetic Irradiation) and Small Sample Pretreatment Methods

Sulfur in organic compounds is completely decomposed in the form of an inorganic substance, and electromagnetic radiation and acid decomposition are simultaneously processed for sufficient chemical equilibrium with concentrated isotopes added as an internal standard solution. The method of acid decomposition through the electromagnetic irradiation used in the experiments of Examples 1 to 4 was taken in a 100 mL sealed Teflon vessel and placed in an electromagnetic oven and heated to 200 ° C. for 15 minutes, and 20 It is to carry out electromagnetic-assisted acid decomposition by keeping 200 degreeC for minutes. The effectiveness of the sample pretreatment method used was intended by comparing the sulfur content in the NIST SRM 2389a (amino acid mixture certified standard material certified by general organic compound purity analysis) with the certified value. 0.2 g of the sample was taken in a Teflon-based container, and the concentrated isotope ³⁴ S (ORNL) was diluted to a pre-calculated concentration and added to the vessel, followed by 3 mL of 65% nitric acid, 3 mL of deionized water, and 2 mL of 30% hydrogen peroxide solution. Acid decomposition was performed. Also, the pretreatment method for acid decomposition of small samples was analyzed for effectiveness by analyzing NIST SRM 2389a samples. 0.2 g of the sample was taken in a small sample-only Teflon container and the concentrated isotope ³⁴ S (ORNL) was added to the vessel after diluting to a pre-calculated concentration, 3 mL of 65% nitric acid was added and placed in an electromagnetic oven for 120 minutes for 8 minutes. Raised up to, and then raised to 220 ℃ for 7 minutes and maintained at 220 ℃ 20 minutes to perform electromagnetic-assisted acid decomposition. NIST SRM2 389a sample is an amino acid standard solution containing mg / kg of methionine (0.3733 ± 0.0108) and mg / kg of cysteine (0.2954 ± 0.0133). Since methionine has one sulfur atom and cysteine has two sulfur atoms, the NIST SRM 2389a sample contains (158.4 ± 4.2) mg / kg. The sulfur content measurement results of the NIST SRM 2389a sample are shown in Table 6 and FIG. 11. It can be seen that the sulfur content measured in both the acid-decomposed sample and the small amount of sample decomposed by the sample pretreatment method used in Examples 1 to 4 is consistent with the certification value of NIST SRM 2389a within the uncertainty range. .

Table 6. Sulfur measurement results of NIST SRM2389a standard amino acid solution.

Example 6 Certification (Secondary Batch) of Sulfur Element-Based Phosphorus Growth Hormone Protein

Phosphorus growth hormone protein certification was performed on freshly prepared hGH secondary batches.

(1) Preparation of Mixed Solution and Calibration Solution

① For the application of isotope dilution mass spectrometry, an isotope ³⁴ S concentrated standard solution (Oak Ridge National Lab.) Was prepared by diluting to a calculated concentration of 3.99 mg / kg. 1 g of each of 11 teflon vessels (sample blend solution) and 4 LDPE bottles (calibration blend solution) were added. When the concentrated isotope solution was added, it was first added to two calibration solution bottles, and then added to 11 sample mixture solution vessels, and finally to the remaining two calibration solution bottles. At this time, ³⁴ S concentrated standard solution (Oak Ridge National Lab.) Was added to prepare a sample blend having an expected isotope ratio ( ³⁴ S / ³² S) of 1.

 ② The samples were collected in 11 microwave digestion teflon vessels each containing 0.2 g each from 11 vials of hGH standard solution. (11 sample blends)

③ The sulfur standard solution (NIST SRM 3154) to be used in the calibration solution was prepared by diluting to 4.12 mg / kg, and then mixed into 1 g of 4 LDPE bottles prepared in ① containing 1 g of ³⁴ S concentrated standard solution. This was diluted to 30 g with 5% nitric acid to prepare four calibration solutions for the expected isotope ratio 1.

 ④ Add 3 mL of 65% sub-boiled nitric acid to the vessel containing the sample blend.

 ⑤ Close the lid of the TFM vessel, heat it up to 120 ° C for 8 minutes in Milestone's UltraWAVE Microwave Digestion System, raise it to 220 ° C for 7 minutes, and hold at 220 ° C for 20 minutes to perform MW-assisted acid decomposition. It was.

 ⑥ After acid decomposition, the vessel was cooled to some extent, and then the sample blend solution was recovered and diluted to 30 g.

(2) ICP / MS measurement

Agilent's 8800 ICP-Triple Quad (ICP-QQQ) was optimized for O ₂ mode. (Filtering m / z = 32, 34 at quadrupole 1 for ³² S and ³⁴ S and reacting with O ₂ gas, m / z = 48, 50 with ³² S ¹⁶ O ⁺ and ³⁴ S ¹⁶ O ⁺ in quadrupole 2 after reaction with O ₂ gas. detection by filtering.)

② The collected sample was added with 20 mL of 5% nitric acid (diluted 17 times) to measure the isotope ratio. (Signal intensity is about 250,000 counts for ³² S and ³⁴ S, respectively.) When measuring the isotope ratio, the isotopic ratio of the known isotope ratio standard sample is measured together with the mass bias correction and bracketing correction of the drift. Corrected.

 ③ Each sample was measured three times, and the measured uncertainty was calculated using the measured isotope ratio, and the uncertainty was estimated by combining the uncertainty evaluation results for each factor affecting the measurement.

(3) Result of hGH secondary batch sulfur content certification by ID-ICP / MS

The second batch of hGH had a manufacturer-supplied concentration of 2 mg / mL and an amino acid based quantitative value of 1.8 mg / mL corresponding to 18.2 mg / kg sulfur content.

 ① The reference value of the measurement of sulfur content of the second batch of hGH is the average value of the measurement results of samples taken from 11 vials, and the homogeneity is estimated from the standard deviation of the measurement values for each of 11 samples. The uncertainty of measurement is the combined standard uncertainty and the degree of freedom from the pooled standard deviation value, which mainly includes the standard deviation of the mean value from 11 samples and the uncertainty due to systematic effects in the sample measurement. freedom was calculated, and the coverage factor and expanded uncertainty were calculated. The standard deviation of the measured values for each bottle is 0.98% of the mean value, as shown in Table 7 and FIG. It can be seen that the homogeneity is suitable for use in the certified standard material within. The certified S content and expansion uncertainty in the secondary batch hGH is mg / kg (18.88 ± 0.75) as indicated in Table 7. Comparing the amino acid and peptide-based quantitative results, it can be confirmed that the authentication values are well matched within the uncertainty range. (See FIG. 13)

Table 7. Sulfur measurement-based certification results for the second batch of phosphorus growth hormone standard solution.

Claims (7)

1. Method for quantifying sulfur-containing organic compounds using sulfur isotope ratio.
2. The method of claim 1,

   A method for quantifying an organic compound containing sulfur, comprising the following steps (1) to (4).

   (1) A sample solution containing an organic compound containing sulfur is diluted with an internal standard solution in which any one isotope selected from ³³ S, ³⁴ S and ³⁶ S is concentrated, and the reference isotope having the greatest natural abundance ratio ( ³² S) Preparing a sample mixture solution such that a theoretical isotope ratio with is a value of 0.2 to 5;

   (2) mixing the sample prepared in step (1) by mixing the internal standard solution in which one isotope selected from ³³ S, ³⁴ S and ³⁶ S is concentrated with the sulfur standard solution having a natural existence ratio Preparing a calibration solution to be similar to the solution;

   (3) recovering sulfur oxides in inorganic form by decomposing organic compounds containing sulfur in the mixed solution prepared in step (1); And

   (4) measuring the isotope ratio of any one isotope and ³² S selected from ³³ S, ³⁴ S and ³⁶ S of the sulfur oxide recovered in step (3), Measuring the corresponding isotope ratio of the calibration solution prepared in step (2).
3. The method of claim 1,

   The sulfur-containing organic compound is a method for quantifying sulfur-containing organic compounds, characterized in that the protein or peptide containing methionine, cysteine or both.
4. The method of claim 1,

   In the step (3), the sulfur-containing organic compound quantitative method characterized in that the simultaneous treatment of electromagnetic radiation and acid decomposition in the mixed solution containing sulfur-containing organic compounds to decompose into sulfur oxides of the inorganic form.
5. The method of claim 4, wherein

   Simultaneous treatment of the electromagnetic wave irradiation and acid decomposition is a method of quantifying sulfur-containing organic compounds, characterized in that the acid-decomposed while irradiating electromagnetic waves at 100 to 300 ℃ temperature conditions after adding an oxidizing agent to the sulfur-containing organic compound.
6. The method of claim 5,

   Wherein the oxidizing agent is nitric acid, perchloric acid, hydrogen peroxide or a mixture thereof, characterized in that the method of quantitative sulfur containing organic compounds.
7. The method of claim 1,

   Any one of the isotope / ³² S isotope ratio and the calibration solution with a ³³ S, ³⁴ S and ³⁶ S any one of isotopes selected from the group consisting of selected from the ³³ S, ³⁴ S and ³⁶ S for the mixed solution obtained in the above step (4) A method for quantifying an organic compound containing sulfur, characterized in that the element / ³² S isotope ratio is applied to <Formula 1>.

   <Equation 1>

   

   C _x : concentration of the sample solution (x) to be measured,

   m _x : mass of the sample solution (x) measured using a balance,

   m _y : Mass of the internal standard solution (y) in which any one isotope selected from ³³ S, ³⁴ S and ³⁶ S added to the sample solution (x) is concentrated

   m _y ': Mass of the internal standard solution (y) in which any one isotope selected from ³³ S, ³⁴ S and ³⁶ S is added to the calibration sulfur standard solution (z)

   C _z : Concentration of calibration sulfur standard solution (z)

   R _x : Sulfur isotope ratio present in sample solution (x) (any one isotope selected from ³³ S, ³⁴ S and ³⁶ S / ³² S isotope)

   R _y: ³³ S, ³⁴ S and ³⁶ S jungeseon selected isotope concentration of internal standard solution (y) the presence of sulfur isotope ratio for the ⁽³³ S, ³⁴ S and ³⁶ S any one of the isotope / ³² S isotope selected from )

   R _z : Isotope ratio of sulfur in sulfur standard solution (z) (any isotope selected from ³³ S, ³⁴ S and ³⁶ S / ³² S isotope)

   R _xi : I-isotope ratio of sulfur in sample solution (x) ( ³² S / ³² S, ³³ S / ³² S, ³⁴ S / ³² S, ³⁶ S / ³² S)

   R _zi : i-isotopic ratio of sulfur present in the calibration sulfur standard solution (z) ( ³² S / ³² S, ³³ S / ³² S, ³⁴ S / ³² S or ³⁶ S / ³² S)

   R _b : Sulfur isotope ratio of the sample mixture solution (any one isotope selected from ³³ S, ³⁴ S and ³⁶ S / ³² S isotope)

   R _{b '} : Sulfur isotope ratio of the correction solution (any one isotopically selected from ³³ S, ³⁴ S and ³⁶ S / ³² S isotope)

   w: dry mass correction factor

   C _blank : concentration of _blank sample

Images