KR20180100915A - Analytic method of fatty acids with improved sensitivity and specificity - Google Patents

Abstract

The present invention relates to a fatty acid quantifying method, and more specifically, to an improved method for simultaneously quantifying fatty acids in a driven blood spot (DBS) with LC/ESI-MS/MS by picolylamine derivatization. An analysis method according to the present invention is advantageous in that storage and sample stability are superior than the conventional blood specimen by using the DBS as a specimen to be analyzed. Also, the analysis method of the present invention is advantageous as compared with the conventional analysis method using blood in that using the DBS as a specimen causes less stress than blood collection and enables easy sample collection by an ordinary person, thereby being used as a point-of-care test kit. In addition, it is possible to simultaneously quantify fatty acids in DBS with LC/ESI-MS/MS by picolylamine derivatization, and to have not only higher sensitivity and specificity but also reproducibility and repeatability than the conventional non-derivatization method.

Description

[0001] The present invention relates to a method for analyzing fatty acids having improved sensitivity and specificity,

The present invention relates to a method for quantifying fatty acids, and more particularly, to an improved method for quantifying fatty acids in dried blood spot (DBS) by simultaneous quantification with picolylamine and LC / ESI-MS / MS ≪ / RTI >

Fatty acids, citric acid intermediates, carbohydrates, amino acids, choline and bile acids are known to be biomarkers of obesity and type 2 diabetes through metabolism. Among these fatty acids, fatty acids are present in the form of free fatty acids, ester forms, or triglycerides and phospholipids in living cells and body fluids, and are closely involved in the regulation of metabolic pathways, cardiovascular and immune system regulation, and brain function development.

In addition, fatty acids have been identified as essential fatty acid deficiencies, fatty acid metabolism disorders, diabetes, obesity, cushing syndrome, hypertrophy, hyperthyroidism, pheochromocytoma, lipodystrophy, hepatitis, liver cirrhosis, TSH hypertrophy, peroxisomal disorder It is also one of the most important factors for diagnosis. As such, fatty acid is one of the most important factors for its accurate analysis in physiological clinic.

However, for the present fatty acid analysis, there is almost no way to analyze the fatty acid in the blood sample obtained by direct blood sampling. However, the numerical value of the specific fatty acid can be changed under a slight stress condition such as blood sampling, Is required.

Meanwhile, the extraction of lipids in the blood can be carried out by, for example, bligh and dyer method, adding a mixed solvent, stirring and then adding a solvent to adjust the solvent ratio to a specific ratio, and then, (Non-Patent Document 1), a method of analyzing the fatty acid composition through the Lepage and Roy method or its modification (Non-Patent Document 2 And 3) are known. However, since the above methods have to be analyzed by collecting blood, there are limitations in that there is a limited route for obtaining analytical samples, and there are objects that are too stressful or difficult to collect at the time of collection.

Recently, there has been much research in the field of basic and clinical researches because it is not stressful and simple compared to blood sampling and can be easily collected even in general households by using DBS (dried blood spot) method.

Currently, fatty acid analysis to determine the fatty acid balance required for metabolism in clinical laboratories and hospitals is mostly based on gas chromatography mass spectrometry (GC-MS). However, this method has a drawback in that accurate quantitative analysis is difficult because it is applied to clinical measurement by measuring the ratio of each fatty acid rather than actual quantification.

In recent years, quantitation has been performed by chromatography / mass spectrometry using various derivatization methods, especially LC / ESI-MS / MS.

On the other hand, with respect to the analysis method using the blood space, a sensitive and specific method (US Pat. No. 9,234,894) for analyzing the adenosine diaminase (ADA) metabolite from the blood reservoir and a method for quantitatively measuring the hexose monophosphate from a biological sample such as blood- (U.S. Patent No. 6,451,611) and the like are known. However, the sensitivity and specificity of the method for analyzing fatty acids by using the hemoglobin as a sample is not known.

In relation to the fatty acid analysis method, techniques such as those used in medical analysis using a solubilizing compound for solubilizing and removing fatty acids (International Publication No. WO 2011/160857) include other chelating agents and antioxidants, A technique for stabilizing fatty acids present in body fluid samples such as blood using a solid medium containing contaminants capable of stabilizing the fatty acids applied to the medium (International Publication No. WO 2013/104025) is known.

However, it is not yet known how to detect fatty acids in blood fats as high sensitivity and high specificity, and methods capable of quantitative analysis of fatty acids even when blood flock is used as a sample.

In addition, it is important to develop a detection method that can increase the quantitative sensitivity because the concentration of fatty acid is lower than that of ordinary liquid blood. In order to accurately quantify trace amounts of fatty acids in the bloodstream, it is necessary to develop a quantitative method that minimizes the matrix effect.

U.S. Patent No. 9,234,894 (registered Jan. 12, 2016) U.S. Patent No. 6,451,611 (registered on September 17, 2002) International Publication No. WO 2011/160857 (published Dec. 29, 2011) International Publication No. WO 2013/104025 (published on Mar. 7, 2013) US Patent 6,800,489 (registered on October 5, 2004)

1. Kim, Jung-Sun, "Effects of Unsaturated Fatty Acid Types and Breeding Duration on Anti-Thrombotic Activity, Blood Composition and Platelet Fatty Acid Composition in Rats", Ewha Womans University Thesis (MA), 1991 2. Lepage G, Roy CC "Direct transesterification of all classes of lipids in a one-step reaction", J. Lipid Res. 1986, 27 (1): 114-120 3. Kim, JS, et al., "Correlation between Serum Cholesterol Levels, Fatty Acid Intake Patterns, Blood Lipids and Fatty Acid Composition in Adult Men and Women", Korean Society of Nutrition, Vol.8, No.2, 2003, 192-201 4. Luginbuhl M et al. "Determination of fatty acid ethyl esters in dried blood spots by LC-MS / MS as markers for ethanol intake: application in a drinking study" Anal Bioanal Chem. 2016 May; 408 (13): 3503-9. 5. Metherel AH et al. "Butylated hydroxytoluene can protect polyunsaturated fatty acids in dried blood spots from degradation for up to 8 weeks at room temperature." Lipids Health Dis. 2013 Feb 20; 12:22. 6. Michel Wagner et al. &Quot; The use of mass spectrometry to analyze dried blood spots " Mass Spectrometry Reviews, 2016, Vol. 35, Issue 3, 361-438. 7. Grace Mashavave, "Determination of omega-3 long chain polyunsaturated fatty acid levels using dried blood spots in Zimbabwean children aged 7 to 9 years", 2013

In the present invention, a new method for quantitatively analyzing fatty acids in dried blood spots (DBS) as a sample is provided.

More specifically, in the present invention, a fatty acid in the blood-bearing tissue, specifically C14 to C26, more specifically C14, C16, C16: 1, C18, C18: 1, C18: 2, C20, C20: : Pycolylamine derivatization of the fatty acids of 5, C22, C22: 1, C22: 6, C24, C24: 1 and C26 and simultaneous quantification with a chromatography / mass spectrometer, specifically LC / ESI-MS / MS I want to provide a way to do it.

In the present invention, there is provided a fatty acid quantitation method which has high sensitivity and high specificity as well as excellent reproducibility and repeatability when compared with the conventional non-lipidated lipid body.

The present invention solves the above problem by providing an analytical method capable of qualitative and quantitative analysis of a fatty acid in dried blood spots (DBS) by derivatizing a picolylamine with a chromatography / mass spectrometer . It has been confirmed that the analytical method according to the present invention has a high sensitivity and specificity when compared with the conventional non-lipidated system.

In one aspect of the present invention, there is provided a method for preparing a compound of formula (I), comprising the steps of: (a) adding picolylamine to dried blood spots (DBS) And (b) analyzing the sample obtained in the step (a). The present invention also provides a method for qualitative or quantitative analysis of fatty acids from hemorrhoids.

In one embodiment of the present invention, the blood space in the step (a) is obtained by putting collected blood cells in a solvent and incubating.

In one embodiment of the present invention, in step (a), the internal standard material before the picolylamine addition is at least one selected from the group consisting of C20-d3, C22-d3, C24-d4 and C26- The method may further comprise the step of adding a substance or a mixture made up of the combination.

Specifically, a standard hemofilter, a hemofilter sample, internal standard materials may be put into a solvent and incubated at a temperature of about 90 ° C to 100 ° C for 1 hour to 2 hours.

Thereafter, it is cooled at room temperature, and a solvent (for example, hexane) is added and mixed. The mixture is centrifuged, and the upper layer is taken out and dried. Then, triphenylphosphine, 2,2'-dipyridyl disulfide, Amine is added, followed by mixing and incubation at about 60 ° C for about 10 minutes to perform the picolylamine derivatization reaction of the fatty acid.

Here, the structure of 2-picolylamine is as shown in the following chemical formula (1). The yield of the final product is varied depending on the structure of the derivatized material during the chemical reaction. In the present invention, when 2-picolylamine was used rather than 3-picolylamine, the yield was higher, and 2-picolylamine was used to derivatize the fatty acid.

≪ Formula 1 >

The picolylamine derivatization reaction of the fatty acid can be carried out as shown in the following reaction formula (1).

<Reaction Scheme 1>

In one embodiment of the present invention, the fatty acid-picolylamine derivative produced through the process as shown in Reaction Scheme 1 can be analyzed by LC / MS by the following formula (2).

(2)

In one aspect of the invention, the fatty acid is, for example, a C14 to C26 fatty acid, and more specifically the fatty acid is selected from the group consisting of myristic acid (C14), palmitic acid (C16), palmitoleic acid , Stearic acid (C18), oleic acid (C18: 1), linoleic acid (C18: 2), arachidic acid (C20), cis- Cis-11-eicosenoic acid, C20: 1, arachidonic acid, C20: 4, cis-5,8,11,14,17-eicosapentaenoic acid, 11,14,17-eicosapentaenoic acid, C20: 5), behenic acid (C22), erucic acid (C22: 1), cis-4,7,10,13,16,19- (Cis-4,7,10,13,16,19-docosahexaenoic acid, C22: 6), lignoceric acid (C24), nervonic acid (C24: 1), and Cerotic acid (C26), and the like.

In one embodiment of the invention, the analysis in step (b) above can be carried out with a chromatography / mass spectrometer, more specifically LC / ESI-MS / MS, whereby simultaneous quantitative analysis of fatty acids in the blood- .

In one embodiment of the present invention, the fatty acid can be quantitatively analyzed even when it is present in the sample in an amount of 500 μg / mL or less, more specifically, the fatty acid is a fatty acid in a range of 1 μg / mL to 500 μg / mL It is applicable to simultaneous quantitative test.

In one aspect of the present invention, the analytical method can be used to determine the specificity, linearity, accuracy, stability, sensitivity, matrix effect, and recovery , And more precisely, precision, accuracy and linearity were evaluated as 80 ~ 120% and accuracy was within 15% (LLOQ: 20%). As a result of the stability test, it was confirmed that the pretreated samples showed no significant change in fatty acid (C14 ~ C26) contents at 10 ℃ for 24 hours. Sensitivity test results of the lowest quantitative limits were also evaluated in accordance with the evaluation criteria. From the above results, it was found that the analysis method according to the present invention is a very accurate and useful analysis method for the simultaneous quantitative determination of fatty acids in blood fats (C14 ~ C26).

The analysis method according to the present invention has an advantage that storage and sample stability are superior to conventional blood specimens by using the blood space as a sample to be analyzed. In addition, since it is less stressful than blood sampling and can be easily sampled by the general public, it is advantageous as a point-of-care test kit, which is advantageous compared with the conventional blood analysis method.

The analytical method according to the present invention is also capable of simultaneous quantification by LC / ESI-MS / MS by using a picolylamine derivative as a fatty acid in the blood-bearing tissue, and has high sensitivity and high specificity as compared with the conventional non- And has an advantageous effect of being excellent in repeatability.

Figure 1 shows the results of fatty acid chromatograms in a normal hematocrit (A: PA-derivatization, B: non-derivatization).

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that the following examples are for the purpose of promoting understanding of the present invention and are not intended to limit the scope of the present invention thereto.

Example 1. Analysis of fatty acid (C20 to C26) profile using LC-MS / MS

<Reagent>

(1) Standard

- arachidic acid (C20) (Sigma, A3631) / frozen (-20 ℃)

Cis-11-eicosenoic acid (C20: 1) (Fluka, 44878) / frozen (-20 ° C)

- Arachidonic acid (C20: 4) (Sigma, 10931) / Frozen (-20 ℃)

-Cis-5,8,11,14,17-eicosapentaenoic acid (C20: 5) (Fluka, 44864) / frozen (-20 ° C)

- Behenic acid (C22) (Fluka, 11909) / Frozen (-20 ℃)

Erucic acid (C22: 1) (Fluka, 45629) / frozen (-20 占 폚)

-Cis-4,7,10,13,16,19-docosahexaenoic acid (C22: 6) (Fluka, 53171)

- lignoceric acid (C24) (Sigma, L6641)

- nervonic acid (C24: 1) (Fluka, 87117)

- Cerotic acid (C26) (Sigma, H0388)

(2) Internal standard

(C20-D3) (C.N.D. Isotope, D-5254)

-Docosanoic acid-22,22,22-d3 acid (C22-D3) (C.N.D. Isotope, D-5708)

- tetracosanoyl-9,9,10,10-d4 acid (C24-D4) (C.N.D. Isotope, D-6167)

(C2D-4) (C.N.D. Isotope, D-6145)

(3) Solvent and Reactant

- Acetonitrile (Fisher Scientific)

- Water (Fisher Scientific)

- methanol (Fisher Scientific)

- 2,2'-dipyridyldisulfide (DPDS) (Sigma, D5767)

- Ammonium acetate (Sigma Aldrich, A7330)

Triphenylphosphine (TPP) (Aldrich, T84409)

<Preparation of DBS calibrator>

Fatty acid-free serum albumin was dissolved in 4% PBS, mixed with standard, and 100 μL was added to Watson 903 paper.

<Major Equipment>

Tandem Mass Spectrometry (model: API 4000 Qtrap, Applied Biosystems, USA)

HPLC system (model: Agilent 1290 Infinity series, USA)

<Sample preparation>

1) The DBS 1 hole was transferred to the glass cap tube using a 3 mm punch.

2) Add 360 μL of acetonitrile and 10 μL of IS mixture (50 mg / mL) to the glass cap tube and mix.

3) Incubated at 90 DEG C for 2 hours.

4) After cooling to room temperature, 2 mL of hexane was added for extraction, and the supernatant was transferred to a new glass cap tube.

5) evaporated under a nitrogen stream.

6) 10 μL TPP (10 mM), 10 μL DPDS (10 mM) and 10 μL PA (10 mg / mL) were added and incubated at 60 ° C. for 10 minutes.

7) evaporated under a nitrogen stream.

8) Dissolve in 1 mL of methanol and inject 2 μL into LC-MS / MS.

<Inspection method>

1) 1.0, 2.5, 5.0, 25.0, 50.0, 100.0, 500.0 mg / mL A standard blood sample (DBS) and a blood sample were punched into a 3 mm pouch and placed in a glass cap tube. C20-d3, C22-d3, C24-d4, C26-d4 mixture 50 mg / mL, in MeOH). 360 μL of acetonitrile and 40 μL of 5 N HCl (in DW) were added and mixed.

2) Incubated at 90 DEG C for 2 hours.

3) After cooling at room temperature, 2 mL of hexane was added and mixed.

4) After centrifuging at 4,000 rpm for 5 minutes, the upper layer was transferred to a new glass cap tube and then evaporated to dryness under a nitrogen stream.

5) 10 μL of triphenylphosphine (TPP, 10 mM), 10 μL of 2,2'-dipyridyl disulfide (DPD, 10 mM) and 10 μL of 2-picolylamine (PA, 10 mg / mL) And then mixed.

6) Incubation was carried out at 60 DEG C for 10 minutes.

7) After cooling at room temperature, it was evaporated to dryness under a nitrogen stream.

8) dissolved in 1 mL of methanol.

9) 2 μL was injected into the LC-MS / MS.

10) The device conditions are shown in Table 1 (HPLC conditions) and Table 2 (ESI-MS / MS (API4000Qtrap) conditions).

11) The results of the experiment are shown in the following Table 3 (DBS analysis result of healthy subjects (unit: [μg / mL])).

Example 2. Validation of the method according to the invention ( validation of the tandem mass spectrometry method for determination of fatty acid (C20 to C26) profile)

This experiment was carried out in ESCEL Healthcare Co., Ltd. for the purpose of verification of the preclinical test for performing fatty acid (C20 ~ C26) test in DBS in relation to the clinical test as a validation test to prove that the measurement result is reliable .

The validity of this study is based on the specificity, linearity, accuracy, precision, stability, sensitivity, matrix effect and recovery rate of the sample Recovery. The results are shown in the following Tables 4 to 13, and the evaluation test results are summarized in Table 14.

Table 4: Summary of C20 validation

Table 5: Summary of C20: 1 validation

Table 6: Summary of C20: 4 validation

Table 7: Summary of C20: 5 validation

Table 8: Validation summary for C22

Table 9: Summary of C22: 1 validation

Table 10: Summary of C22: 6 validation

Table 11: Summary of C24 validation

Table 12: Summary of C24: 1 validation

Table 13: Validation summary for C26

Table 14: Summary of experiment results

As a result of the test, all evaluation items in this validation evaluation test passed the test standard. The accuracy, accuracy and linearity of day and day were verified as 80 ~ 120% and 15% (LLOQ: 20%), respectively. As a result of the autosampler stability test, the pretreated samples showed no significant change in the fatty acid (C20 ~ C26) content at 10 ℃ for 24 hours. The sensitivity test of the lowest quantitative limit value was evaluated in accordance with the evaluation criteria. Matrix effect test showed 20.50 ~ 58.98% as a whole.

From the above results, it can be understood that the test method according to the present invention can be applied to the simultaneous quantitative test of 1.0 to 500 μg / mL of C20 to C26 in DBS.

The results of the experiment are summarized in the following Table 14:

Summary of experiment results Linearity The linearity of C20, C20: 1, C20: 4, C20: 5, C22, C22: 1, C22: 6, C24, C24: Accuracy evaluation criteria: Within 85 ~ 115% (LLOQ: Within 80 ~ 120%) Fits standards Precision evaluation criteria: Within 15% (LLOQ: within 20%) Intra-day Accuracy and Precision Accuracy and precision in the daytime of C20, C20: 1, C20: 4, C20: 5, C22, C22: 1, C22: 6, C24, C24: Accuracy criteria: 85 to 115% and precision criteria: less than 15% Fits standards Inter-day Accuracy and Precision Accuracy and Precision of Daily Accuracy of C20, C20: 1, C20: 4, C20: 5, C22, C22: 1, C22: 6, C24, C24: Accuracy criteria: 85 to 115% and precision criteria: less than 15% Fits standards Specificity
(Specificity) Specificity of C20, C20: 1, C20: 4, C20: 5, C22, C22: 1, C22: 6, C24, C24: Identification of separation from other components in the sample Fits standards Stability - Autosampler stability (10 ℃) The autosampler stability of C20, C20: 1, C20: 4, C20: 5, C22, C22: 1, C22: 6, C24, C24: Evaluation after 24 hours after mounting on Autosampler Fits standards Accuracy criteria: 85 to 115% and precision criteria: less than 15% Sensitivity of the LLOQ value Sensitivity of the lowest quantitative limit value of C20, C20: 1, C20: 4, C20: 5, C22, C22: 1, C22: 6, C24, C24: Accuracy evaluation criteria: Within 80 ~ 120% Fits standards Precision rating criteria: within 20% Matrix effect
(Matrix effect) Matrix effect of C20, C20: 1, C20: 4, C20: 5, C22, C22: 1, C22: 6, C24, Recovery rate
(Recovery) Recovery of C20, C20: 1, C20: 4, C20: 5, C22, C22: 1, C22: 6, C24, C24:

<Abbreviation explanation>

FA: fatty acid (fatty acid)

PA: picolylamine (picolylamine)

LC: liquid chromatography

MS: mass spectrometry (mass spectrometer)

ESI: electrospray ionization (electrospray ionization)

DBS: dried blood spot

Claims (10)

(a) adding picolylamine to dried blood spots (DBS) to react; And
(b) analyzing the sample obtained in step (a).
Methods for the qualitative or quantitative analysis of fatty acids from blood reservoirs.
The method according to claim 1,
Wherein the picolylamine is 2-picolylamine represented by the following formula (1): < EMI ID =
&Lt; Formula 1 &gt;

.
The method according to claim 1,
Wherein the blood space in the step (a) is obtained by inserting the collected blood blank in a solvent.
The method according to claim 1,
In the step (a), as the internal standard material before the picolylamine addition, a substance or a mixture made of at least one selected from the group consisting of C20-d3, C22-d3, C24-d4 and C26- &Lt; / RTI &gt; further comprising the step of adding a &lt; RTI ID = 0.0 &gt;
The method according to claim 1,
Fatty acids include myristic acid (C14), palmitic acid (C16), palmitoleic acid (C16: 1), stearic acid (C18), oleic acid : 1), linoleic acid (C18: 2), arachidic acid (C20), cis-11-eicosenoic acid (C20: 1), arachidonic acid C20: 4), cis-5,8,11,14,17-eicosapentaenoic acid (Cis-5,8,11,14,17-eicosapentaenoic acid, C20: 5), behenic acid ), Erucic acid (C22: 1), cis-4,7,10,13,16,19-docosahexanoic acid (cis-4,7,10,13,16,19- docosahex- wherein the fatty acid is at least one selected from the group consisting of lignoceric acid (C22: 6 aenoic acid, C22: lignoceric acid, C24: nervonic acid, Qualitative or quantitative analysis methods.
The method according to claim 1,
Wherein the analysis in step (b) is a chromatography / mass spectrometry.
Wherein the analysis in step (b) is LC / ESI-MS / MS.
The method according to claim 1,
Fatty acid can be quantitatively analyzed even when it is present in the sample in an amount of less than 500 μg / mL.
The method according to claim 1,
Fatty acid is a fatty acid qualitative or quantitative analysis method applicable to simultaneous quantitative test of fatty acid in blood flesh in a range of 1 μg / mL to 500 μg / mL.
The method according to claim 1,
The analysis method may include one or more of evaluation items consisting of items of specificity, linearity, accuracy, stability, sensitivity, matrix effect, and recovery / RTI &gt; acid or a pharmaceutically acceptable salt thereof.

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