Patent Application
20140315322
The present application is filed claiming the priority of the Japanese Patent Application No. 2011-287835 (Filing date: Dec. 28, 2011), the entire contents of which are herein incorporated by reference.
The present invention relates to a simple and highly reproducible method for measuring the concentration of acetic acid in blood plasma (particularly, human blood plasma) by using a gas chromatography/mass spectrometry (GC/MS method).
Among blood purification methods applicable to chronic renal failure patients and the like, the most common one is hemodialysis treatment. Hemodialysis treatment is used for removing waste products or water in blood, and correcting the concentration of serum electrolytes, correcting acid-base balance, etc. by dialysis agents.
Conventionally, acetate dialysis using acetate as an alkaline agent in dialysis fluid has been used. Nowadays, however, bicarbonate dialysis using sodium hydrogen carbonate as an alkaline agent is mainly used because it has been found that acetic acid exerts action on vasodilatation or inhibitory heart function. In bicarbonate dialysis, 8 to 12 mEq/L of acetic acid is still used as a pH regulator. A sharp rise in the concentration of acetic acid in blood plasma has been reported to lead to severe allergic reaction (Non-Patent Document 1). Accordingly, it is very important to measure the concentration of acetic acid in blood plasma in a patient during dialysis treatment in order to recognize its variation. As of now, however, the concentration of acetic acid in blood plasma is not examined in medical institutions or contract research organizations in Japan.
As methods for measuring the concentration of acetic acid in blood plasma, enzyme methods (Non-Patent Document 2 to 3), HPLC methods (Non-Patent Documents 4 to 6), GC methods (Non-Patent Documents 7 to 9) and GC/MS methods (Non-Patent Documents 10 to 12) have been reported. Among them, GC/MS methods have the highest specificity and sensitivity.
In conventional measurement methods using GC/MS, there is a problem of complicated procedures as follows: the results tend to vary depending on the operation due to using no stable isotope labeled acetic acid as an internal standard (Non-Patent Documents 10 and 12); it is necessary to add hydrochloric acid before extraction; diethyl ether having a low boiling point is used as an extracting solvent; and the like (Non-Patent Documents 11 and 12).
An object of the present invention is to provide a simple and highly reproducible method for measuring the concentration of acetic acid in blood plasma by using a GC/MS method.
The present inventors have studied so as to resolve the above problem and found that the number of operating procedures can be reduced by deproteinization and then liquid-liquid extraction directly without centrifugation after the deproteinization, and the operating procedures can be facilitated without the decrease of extraction efficiency by using, as an extracting solvent, methyl-tert-butyl ether (MTBE) that has a high boiling point, that is easy of handling, and that shows high extraction efficiency, thus leading to the present invention.
The present invention includes the followings:
[1] A method for measuring the concentration of acetic acid in blood plasma by using a gas chromatography/mass spectrometry (GC/MS method), which comprises extracting acetic acid in blood plasma with methyl-tert-butyl ether (MTBE).
[2] The method according to the above [1], which does not comprise adding hydrochloric acid before extracting.
[3] The method according to the above [1] or [2], which further comprises treating blood plasma with a deproteinizing agent.
[4] The method according to any one of the above [1] to [3], wherein the deproteinizing agent includes sulfosalicylic acid.
[5] The method according to the above [3] or [4], which comprises extracting acetic acid in blood plasma with MTBE, after treating blood plasma with a deproteinizing agent and not centrifuging.
[6] The method according to any one of the above [1] to [5], which further comprises adding a stable isotope labeled acetic acid to blood plasma.
[7] The method according to the above [6], wherein the stable isotope labeled acetic acid is selected from sodium acetate-1-13C, sodium acetate-2-13C, sodium acetate-13C2, sodium acetate-d3, sodium acetate-18O2, sodium acetate-1-13C,d3 and sodium acetate-2-13C,d3.
[8] The method according to any one of the above [1] to [7], wherein the GC/MS method includes electron impact ionization.
[9] A kit for measuring the concentration of acetic acid in blood plasma by using a GC/MS method, comprising MTBE.
[10] The kit according to the above [9], which further comprises a deproteinizing agent.
[11] The kit according to the above [10], wherein the deproteinizing agent includes sulfosalicylic acid.
[12] The kit according to any one of the above [9] to [11], which further comprises a stable isotope labeled acetic acid.
[13] The kit according to the above [12], wherein the stable isotope labeled acetic acid is selected from sodium acetate-1-13C, sodium acetate-2-13C, sodium acetate-13C2, sodium acetate-d3, sodium acetate-18O2, sodium acetate-1-13C,d3 and sodium acetate-2-13C,d3.
[14] The kit according to any one of the above [9] to [13], which further comprises a derivatizing reagent.
[15] The kit according to the above [14], wherein the derivatizing reagent is N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide.
According to the present invention, it is possible to measure the concentration of acetic acid in blood plasma in a simple and highly reproducible method due to the improvement of extraction efficiency of acetic acid by using MTBE having a high boiling point as an extracting solvent.
Further, according to the present invention, it is possible to perform solvent extraction directly, without centrifugation after the deprotainization and without addition of hydrochloric acid before the extraction.
The present invention relates to a method for measuring the concentration of acetic acid in blood plasma by using a gas chromatography/mass spectrometry (GC/MS method), which comprises extracting acetic acid in blood plasma with methyl-tert-butyl ether (MTBE) (hereinafter sometimes referred to as “the method of the present invention”).
Blood plasma is one of liquid components of blood, accounts for 55% of blood, and is comprised of blood serum and fibrinogen. It involves in transportation of material, exchange of gas, blood coagulation, or immunity, and plays significant roles to control internal circumstances by adjusting osmotic pressure, hydrogen-ion concentration, etc. The “blood plasma” to be measured by the present invention is not particularly limited, but includes those from blood of mammals such as humans, rats, mice, dogs, monkeys, etc. The production method thereof is also not particularly limited, but may include conventional methods. The blood plasma is preferably prepared by drawing blood, placing the blood into a plastic tube containing an anticoagulant (e.g., heparin sodium, EDTA-2Na, EDTA-2K, etc.), cooling the mixture with ice, and centrifuging the mixture at 4° C. to collect blood plasma.
Gas chromatography/mass spectrometry (GC/MS method) is useful for qualitative or quantitative analysis of organic compounds (particularly, low-molecular-weight components) by an analysis equipment comprising a gas chromatograph (GC) and a mass spectometer (MS) linked together. In the method, qualitative analysis of components is performed by measuring MS spectrum of a single component separated by GC, and quantitative analysis is performed by measuring ion intensity detected by MS. Examples of “gas chromatography/mass spectrometry (GC/MS method)” in the present invention is not particularly limited, but include those generally used for identification, quantitative analysis, etc. of substances, preferably those using electron-impact ionization (EI), those using chemical ionization (CI), and the like (for example, the above Non-Patent Documents 10, 11 and 12).
The method of the present invention comprises extracting acetic acid in blood plasma with methyl-tert-butyl ether (MTBE).
For extracting acetic acid in blood plasma, hexane, diethyl ether, etc. have been conventionally used. It is, however, necessary to carefully handle diethyl ether because of its low boiling point. In addition, there are some problems with these solvents as follows: hexane provides low extraction efficiency of acetic acid; the extraction with diethyl ether requires the addition of a certain amount of hydrochloric acid beforehand (for example, 0.025 to 0.125 mL of concentrated hydrochloric acid (12N) as hydrochloric acid is required relative to 1 mL of a solution after deproteinization). These solvents, therefore, make the operating procedures complicated.
In contrast, MTBE has a boiling point higher than diethyl ether and provides higher extraction efficiency of acetic acid than diethyl ether or hexane, and thus does not cause the above problems with conventional extracting solvents.
In the present invention, the amount of MTBE to be used can be determined based on the amount of blood plasma, and it is generally 0.004 to 40 mL, preferably 0.04 to 4 mL, relative to 0.4 mL of blood plasma.
In general, the above extraction with MTBE is carried out as follows. Firstly, a certain amount of MTBE is mixed to blood plasma, and then the mixture is stirred for a certain time. The stirring time and temperature are not particularly limited, but the stirring time may include, for example, 30 seconds to 1 minute and the temperature may include, for example, 15 to 30° C. After that, the mixture is centrifuged to remove a precipitate and to collect a supernatant. The centrifugation may be carried out, for example, at 10000 rpm at 4 to 20° C. for 5 to 10 minutes.
As a result of the above procedures, acetic acid in blood plasma is extracted into a supernatant (MTBE).
When diethyl ether, which is conventionally used as an extracting solvent, is used for the extraction of acetic acid, it is necessary to add a certain amount of hydrochloric acid to blood plasma beforehand (for example, 0.025 to 0.125 mL of concentrated hydrochloric acid (12N) as hydrochloric acid is required relative to 1 mL of a solution after protein removal). This is because an aqueous solution needs to be acidified when using diethyl ether. From the present comparative investigation, it has, however, been found that the MTBE as an extracting solvent can provide higher extraction efficiency than diethyl ether without requiring the addition of hydrochloric acid. The method of the present invention is thus simpler and achieves a higher yield compared to conventional methods.
The method of the present invention may further comprise treating blood plasma with a deproteinizing agent. Examples of the deproteinizing agent to be used in the method of the present invention include sulfosalicylic acid, perchloric acid, metaphosphoric acid, and the like, preferably sulfosalicylic acid.
By the treatment of blood plasma with the deproteinizing agent, proteins in blood plasma are coagulated and suspended in blood plasma. As a result, proteins in blood plasma are substantially removed (deproteinizing step). Conventionally, proteins suspended in blood plasma have been removed as a precipitate by centrifugation and only supernatant has been used for the measurements by GC/MS methods and the like, because such proteins have been considered to cause adverse effects on the measurements.
The present inventors have, surprisingly, found that the product of deproteinizationcan be directly, without centrifugation, subjected to extraction of acetic acid with MTBE. Namely, no centrifugation after the treatment with the deproteinizing agent is necessary for the present invention, and the number of the operating procedures is thus reduced. If 100 samples are measured by the method of the present invention, it is possible to shorten the time for operating procedures by about 60 minutes compared to conventional methods.
The amount of the deproteinizing agent to be used in the present invention can be determined based on the amount of blood plasma. For example, when 10% sulfosalicylic acid is used as the deproteinizing agent, the amount thereof may be generally 0.001 to 10 mL, preferably 0.01 to 1 mL, relative to 0.4 mL of blood plasma. The concentration of the deproteinizing agent can be arbitrarily varied, thereby changing the yield.
The above treatment with the deproteinizing agent is generally carried out as follows. Firstly, a certain amount of the deproteinizing agent is mixed to blood plasma, and the mixture is stirred for a certain time. The stirring time and temperature are not particularly limited, but the stirring time may include, for example, 30 seconds to 1 minute and the temperature may include, for example, 15 to 30° C. As a result, proteins in blood plasma are coagulated to give a suspension.
In conventional methods, the suspension is subjected to centrifugation to remove suspended proteins as a precipitate. In contrast, in the method of the present invention, it is possible to subject the suspension from the treatment of the deproteinization to the next step (in general, MTBE extraction) without centrifugation
In the method of the present invention, the treatment with the deproteinizing agent is generally performed before the extraction of acetic acid in blood plasma with MTBE. By this step, the interference by proteins can be prevented in the extraction with MTBE.
The method of the present invention may further comprise adding a stable isotope labeled acetic acid to blood plasma. The stable isotope refers to isotopes not releasing radiation and existing semipermanently without changing the abundance thereof. Examples of the stable isotope to be used for labeling include, in addition to stable isotopes of carbon (12C and 13C), stable isotopes of hydrogen (1H and 2H(D)), stable isotopes of oxygen (16O, 17O, and 18O), and the like. The stable isotope labeled acetic acid can be used as an internal standard, thereby correcting the variations of the results depending on the measuring operation.
Examples of the “stable isotope labeled acetic acid” in the present invention include sodium acetate-1-13C, sodium acetate-2-13C, sodium acetate-13C2, sodium acetate-d3, sodium acetate-18O2, sodium acetate-1-13C,d3 and sodium acetate-2-13C,d3, and the like, preferably sodium acetate-2-13C,d3, and the like. They are commercially available, and can be purchased from, for example, ISOTEC, Cambridge Isotope Laboratories, etc.
In the method of the present invention, the addition of the stable isotope labeled acetic acid to blood plasma is generally carried out before the protein removal or MTBE extraction because the internal standard is used for correcting the variations depending on the measuring operation.
In the method of the present invention, a derivatizing reagent is generally added to the sample obtained by the above each step to derivatize acetic acid in the sample, and then the concentration of acetic acid in the sample is measured by the GC/MS method. The derivatizing reagent to be used in the present invention is not particularly limited, but includes, for example, N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBSTFA), t-butyldimethylsilylimidazole (TBDMS), and the like. They are commercially available, and can be purchased from, for example, Sigma-Aldrich, Fulka, Pierce, etc.
The present invention further provides a kit for measuring the concentration of acetic acid in blood plasma, which can be used for the above method of the present invention for measuring the concentration of acetic acid in blood plasma by a GC/MS method.
The kit of the present invention may comprise various reagents, which can be used for the method of the present invention, particularly the kit may one or more reagents such as MTBE, deproteinizing agent, stable isotope labeled acetic acid, derivatizing reagent for GC/MS method, sodium acetate, etc.
Sodium acetate (82 mg, Lot No. MKBD4171V, manufactured by SIGMA ALDRICH) was precisely weighted with an electric balance (AX205DR, manufactured by Mettler Toledo) and dissolved and made up to exactly 10 mL with purified water to prepare a 100 mmol/L solution (S). Further, as shown in Table 1, standard solutions (W1 to W8) were prepared by using a measuring flask before use.
Stable isotope labeled sodium acetate (8.6 mg, Lot No. EK1873: sodium acetate-2-13C, d3, manufactured by ISOTEC) was precisely weighted and dissolved and made up to exactly 10 mL with purified water to prepare a 10 mmol/L solution (IS). Next, 10 mmol/L solution (IS) (2 mL) was taken into a measuring flask, made up to exactly 10 mL with purified water to prepare a 2 mmol/L solution (IS-W1).
As samples for calibration curve, the standard solutions W3 to 8 were used.
Into a polypropylene tube, each sample (400 μL, samples for calibration curve and human blood plasma) was taken, and the internal standard solution IS-W1 (100 μL) was added thereto. Further, 10% sulfosalicylic acid (special grade, manufactured by Wako Pure Chemical Industries) solution (100 μL) was added thereto and stirred. To this solution was added MTBE (400 μL, Lot No. EPF0734, manufactured by Wako Pure Chemical Industries) and stirred for 1 minute. After that, the mixture was subjected to centrifugation (RX-200, manufactured by TOMY SEIKO) at 10000 rpm at 4° C. for 5 minutes, and the supernatant (200 μL) was taken into another glass vial. Then, to the supernatant was added N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) (5 to 10 μL, Lot No. BCBC7878, manufactured by ALDRICH) and heated with Block Heater (DTU-2B, manufactured by TAITEC) at 60° C. for 1 to 2 hours. After that, the resultant solution was measured by using GC/MS (JMS-AMII150, manufactured by JEOL) in the following conditions.
[GC/MS measurement condition]
Samples for calibration curve together with human blood plasma were pretreated and measured in the same method as that of Example 1. Both mass chromatograms were compared and assessed for the presence or absence of interference peaks at the elution position of the internal standard.
Samples for calibration curve (standard solutions W3 to 8) were pretreated and measured in the same method as that of Example 1. The relative error (% RE) in each concentration in the calibration curve and the linearity of the calibration curve were examined.
Next, the peak areas of acetic acid and a stable isotope labeled acetic acid with or without the hydrochloric acid pretreatment were compared. The amount of each sample added is shown below.
The peak areas of the above samples were obtained according to the method described in Example 1. The results are shown in Table 2.
The peak areas of the above samples were obtained according to the method described in Example 1. The results are shown in Table 3.
As shown in Tables 2 and 3, the extraction with MTBE without the addition of hydrochloric acid gave the best result in extraction efficiency in both standard solutions and blood plasma.
Solutions W1 to 8 obtained in “1. Preparation of acetic acid standard solutions” of Example 1 were used.
Solution IS-W1 obtained in “2. Preparation of internal standard solution” of Example 1 was used.
As samples for calibration curve, the standard solutions W3 to 8 were used.
Standard solution S (180 μL) was taken into a 10 mL measuring flask and diluted to 10 mL with human blood plasma to prepare an acetic acid (1800 μmol/L) added blood plasma (P1). Further, as shown below, acetic acid added blood plasmas (P2 to 5) were prepared by using commercial human blood plasma.
Samples were pretreated in the same method as that of “4. Pretreatment method and measurement method for samples” of Example 1, and then measured by GC/MS (5975C GCMSD, manufactured by Agilent) in the following conditions.
Calibration curve used in Example 1 was used.
Solutions W2 to 8 obtained in Example 2 were used.
Solutions P1 to 5 obtained in Example 2 were used.
Blood plasma from patient (n=45) being treated with a dialysis fluid containing acetic acid at 2 and 4 hours after the dialysis initiation were used as the samples.
Each sample (200 μL, samples for calibration curve, human blood plasma, samples of acetic acid-added blood plasma and samples of blood plasma from dialysis patient) was taken into a cuvette, and purified water (800 μL) was added thereto. The measurement was performed in accordance with the method described in the package insert of F-kit acetic acid (Lot/Ch.-B.: 12670700, J. K. international) except for using a half amount of the reagent solution attached to the kit.
[Measurement wavelength]
Wavelength: 340 nm
From ΔE of samples for calibration curve (W2 to 8, n=1), calibration curve (Y=aX+b, Y: ΔE, X: concentration μmol/L) was prepared by using least-squares method. No weighting of calibration curve was carried out. The resultant calibration curve is shown in
The measured value of each sample was calculated by applying the peak area ratio or ΔE to the calibration curve, and rounding off to one decimal place.
RE was calculated according to the following equation:
The measurement results of samples for calibration curve by the GC/MS method (Example 2) and the enzyme method (Comparative Example 3) are shown in the following Tables 4 and 5. Both methods produce a good result.
The measurement results of blood plasma and acetic acid-added blood plasma by the GC/MS method (Example 2) and the enzyme method (Comparative Example 3) are shown in the following Tables 6 and 7. In the GC/MS method, the recovery rate was a good value, 92% or more, at every concentration of the added acetic acid. In the enzyme method, the recovery rate was 90% or less at 50 and 150 μmol/L of the concentration of the added acetic acid.
Comparison of the measured values in both methods is shown in the following Table 8. For every sample, the GC/MS method gave higher value than the enzyme method. Both measured values tended to be closer with increasing the concentration of the added acetic acid.
Table 9 shows measured values by the enzyme method (Comparative Example 3) for blood plasma of dialysis patient (n=45) being treated with an acetic acid-containing dialysis fluid, and measured values by the GC/MS method (Example 2) for the same samples. In 43 cases among 45 cases, measured values by the GC/MS method were higher than those by the enzyme method.
As described above, the GC/MS method (Example 2) has superior sensitivity and specificity compared to the enzyme method (Comparative Example 3). The GC/MS method is thus considered to be useful for measuring the concentration of acetic acid in blood plasma.
Enzyme methods have been reported to be sensitive to interfering substances in blood plasma (see Bergmeyer H U and Mollering H: In: Methods of Enzymatic Analysis (ed by Bergmeyrer H U, et al), 3rd ed, vol VI, p. 628-645, W2-einheim, Deerfield Beach, Fla., Verlag Chimemine, 184. (1985)). The above results implied this matter.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2011-287835 | Dec 2011 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2012/083663 | 12/26/2012 | WO | 00 | 6/19/2014 |
