KR20150109995A - A method for detecting porphyrin in a biospecimen using LC-MS/MS - Google Patents

Abstract

The present invention relates to a method for detecting porphyrin by pretreating biological samples using liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / Ms). The present invention can effectively detect a small amount of porphyrin. Therefore, the method can detect porphyrin using biological samples containing a small amount of porphyrin such as blood, can precisely detect porphyrin in the case of restricting the number of collection of biological samples and an amount of biological samples which can be collected like newborn infants and thus can be useful in diagnosing porphyrin-related diseases in early stages.

Description

The present invention relates to a method for detecting porphyrin in a biological sample using LC-MS / MS,

The present invention relates to a method for detecting porphyrin using LC-MS / MS by pretreating a biological sample.

Porphyrin is a substance that forms a heme together with iron and becomes a component of hemoglobin, hemoglobin. Porphyrin is also a component of cytochrome that plays an important role in the oxidation and reduction reaction in vivo, and is a component of chlorophyll involved in photosynthesis.

Porphyrin is related to various diseases. A typical example is porphyria, which is caused by the accumulation of porphyrin and iron in organs due to deficiency of enzymes involved in the synthesis of heme due to inherited or acquired genetic defects. Symptoms of pityriasis usually include neuromuscular symptoms or skin symptoms. Neuropsychiatric symptoms are manifested after puberty and are more common in women and are associated with symptoms such as acute attacks, abdominal pain, weakened sensory nervous system . Skin symptoms usually appear in their 30s or older, with excessive exfoliation, blisters, scars, and hepatic manifestations after sun exposure. At present, there is no cure for porfyriasis. If you are diagnosed with pityriasis, reduce exposure to sunlight, reduce exposure to drugs, alcohol, female hormones, or periodically slaughter to prevent the accumulation of iron It is known that taking preventive measures such as reducing the symptoms can reduce the symptoms. It is reported that the mortality rate is 10 to 40% if early diagnosis of pityriasis is not made.

Recently, porphyrin has attracted attention as a biomarker of autism (autism spectrum disorder). It has been found that the concentration of porphyrins coproporphyrin, hexacarboxy porphyrin and pentalcarboxyporphyrin is increased in the urine of autistic children. In this regard, there are many reports that porphyrin, which exists in an excessive amount in the body, triggers autism by causing heavy metals such as mercury to stay in the body for a longer time. At present, there is no known treatment method for autism. However, it has been reported that autism is improved by chelation therapy for eliminating heavy metals in the body by administering a chelator such as DMSA or ALA to diagnose autism (Nafta et al. , 2006).

As described above, although it is important to detect and analyze porphyrin and diagnose early if there is a porphyrin-related disease, the porphyrin test developed until now is limited. Porphyrin is released into urine, stools and bile, especially in urine. Porphyrin is also present in the blood, but it is difficult to use for examination because of its small amount. The porphyrin test is carried out using LC-MS / MS. At present, there is a problem that a small amount of porphyrin is difficult to detect.

It is an object of the present invention to provide a method for detecting porphyrin in a biological sample using LC-MS / MS including pre-treating a biological sample with a mixed solution of methanol and strong acid.

The present invention

1) collecting a biological sample from a subject;

2) pre-treating the biological sample with a mixed solution of methanol and strong acid; And

3) A method for detecting porphyrin in a biological sample, comprising the step of analyzing the pretreated biological sample by LC-MS / MS.

In the method of the present invention, the 'object' may be a person.

In the method of the present invention, the 'pre-treatment' in the step 2) refers to contacting or mixing the biological sample with a mixed solution of methanol and strong acid.

In the present invention, 'porphyrin' refers to a compound having the following basic structure and derivatives thereof.

[constitutional formula]

Porphyrins can have various forms, and porphyrins released from blood, urine and stools are typically composed of protoporphyrin, harderoporphyrin, coproporphyrin, pentacarboxylporphyrin, hexacarboxy Hexacarboxylporphyrin, Heptacarboxylporphyrin, and Uroporphyrin. These are collectively referred to as porphyrins. In addition, each porphyrin has I, II, and III types, all of which are also called porphyrins.

Many types of porphyrins contain carboxyl groups. For example, 2 for protoporphyrin, 3 for hadoroporphyrin, 4 for coproporphyrin, 5 for pentacarboxy porphyrin, 6 for hexacarboxy porphyrin, 6 for heptalcarboxy porphyrin, And the europorphyrin contains 8 carboxyl groups. The higher the number of carboxyl groups, the higher the water solubility is discharged into the urine, and the 4-carboxyl group of porphyrin is excreted in both urine and stool. Hetero-porphyrin and protoporphyrin are almost excreted.

LC-MS / MS is a liquid chromatograph coupled to Tandem Mass Spectrometry (Liquid Chromatography) coupled to a liquid chromatograph equipped with a column and HPLC. In LC-MS, samples are injected into a column by HPLC and liquid chromatography separates the materials contained in the sample. Separated materials enter the mass spectrometer and are ionized to show signal peaks at different times, You can see what they are. A plurality of mass spectrometers (usually two) connected LC-MS / MS can more accurately identify the analyte. LC-MS / MS is widely used for the analysis of many materials because of the advantage of separating the material from the sample and allowing accurate identification of the material.

However, compounds containing a large number of carboxyl groups such as porphyrin are very difficult to analyze using LC-MS / MS. This is because ionization is inhibited by the carboxyl group, which is a functional group of porphyrin, when ionized in a mass spectrometer, resulting in a decrease in the mechanical sensitivity and precise analysis. The method of the present invention enables precise detection of porphyrin by esterifying porphyrin by replacing carboxyl groups with methyl ester groups and removing the produced water by pretreatment of a biological sample collected with a mixed solution of methanol and strong acid.

Since the method of the present invention can detect a small amount of porphyrin, it can be more advantageously used when the amount of biological sample to be collected is small. An example of a case where the amount of the biological sample to be collected is small is the case of a newborn baby. Newborns of small size and very sensitive to stimuli have limitations on the number of biopsy samples and the amount of biological sample that can be taken. With the method of the present invention, blood porphyrin can be sufficiently detected by only one drop of blood, that is, about 3 μl of blood. Therefore, in the present invention, the subject may be a newborn within 1 month of birth, preferably within 7 days of birth, more preferably within 72 hours of birth. However, the age of the subject to which the method of the present invention can be applied is not limited thereto.

An example in which the method of the present invention can be utilized is a neonatal screening test. In congenital metabolic disorders, if diagnosis is delayed, it can cause irreversible damage. However, because there are diseases that can prevent such damage if it is diagnosed and treated early, the Government of the Republic of Korea will select 6 new congenital metabolic disorders The majority of parents are screening for dozens of other diseases. The test is performed at 3 to 7 days after birth by collecting blood from the heel of the newborn baby, dropping it on the blood filter paper, drying it, and sending it to the laboratory for analysis. The amount of blood contained in the blood filter paper is only about 3 μl. Since the porphyrin can be analyzed using the blood filter paper according to the present invention, it is possible to detect porphyrin without additionally taking a biological sample.

In the method of the present invention, the porphyrin is preferably selected from the group consisting of porphyrin containing a carboxyl group, more preferably at least four carboxyl groups, such as co-porphyrin, pentacarboxy porphyrin, hexacarboxy porphyrin, Porphyrin. However, the type of porphyrin is not limited thereto.

Using the method of the present invention, porphyrin can be effectively detected even in a biological sample containing a small amount of porphyrin. Therefore, in the method of the present invention, the biological sample of step 1) may preferably be blood, for example, whole blood, plasma, or serum. However, the type of the biological sample is not limited thereto, and urine, stool, and bile may be biological samples.

In the process of the present invention, the strong acid in step 2) may be sulfuric acid, hydrochloric acid or nitric acid. Preferably, the strong acid may be sulfuric acid. However, the type of strong acid is not limited thereto, and other strong acid can be used.

In the method of the present invention, the mixed solution of methanol and strong acid in step 2) may be a mixture of methanol and strong acid at a volume ratio of 9: 1 to 8: 2. Preferably, methanol and strong acid are mixed at a volume ratio of 9: 1. However, the mixing ratio is not limited thereto, and other mixing ratios are possible.

In the method of the present invention, the step 1) may be carried out as a method including the following steps.

a) collecting a biological sample from a subject;

b) dropping the biological sample on a blood filter paper; and

c) eluting the biological sample from the blood filter paper.

In one embodiment of the present invention, the elution of the biological sample in step c) may be performed by contacting formic acid with blood filter paper from which the biological sample has been dropped. When formic acid is used, blood contained in the porous blood filter paper is effectively eluted. A 0.5M dilution of distilled water formic acid can be preferably used. However, the concentration of usable formic acid is not limited thereto.

By using the present invention, a small amount of porphyrin can be detected very effectively. Therefore, it is possible to detect porphyrin even in a biological sample containing a small amount of porphyrin, such as blood, and even when the number of samples of the biological sample and the amount of the biological sample to be collected are very limited Precise detection of porphyrin is possible. Therefore, it can be useful for early diagnosis of porphyrin-related diseases.

1 is a schematic view showing a blood filter paper.
A of Figure 2 depicts the structure of a porphyrin I- ¹⁵ N ₄ to CP, PP, HexaP, HeptaP, UP, and Cope, B shows the structure of the esterified compound.
FIG. 3A shows the result of mass spectrometry of CP, PP, HexaP, HeptaP and UP, and B shows the results of mass spectrometry when the compounds were esterified.
Fig. 4A shows the result of LC-MS / MS analysis of the blank sample, and B shows the result of LC-MS / MS analysis of the 50 nmol / L CPTE solution of CPTE.
Fig. 5A shows the result of LC-MS / MS analysis of the blank sample, and B shows the result of LC-MS / MS analysis of the PPPE 50 nmol / L calibration curve solution.
FIG. 6A shows the result of LC-MS / MS analysis of the blank sample, and B shows the result of LC-MS / MS analysis of the HexaPHE 50 nmol / L calibration curve solution.
Fig. 7A shows the result of LC-MS / MS analysis of the blank sample, and B shows the result of LC-MS / MS analysis of the 50 nmol / L HeptaPHE calibration curve solution.
Fig. 8A shows the result of LC-MS / MS analysis of the blank sample, and B shows the result of LC-MS / MS analysis of the 50 nmol / L UPOE calibration curve solution.

Embodiments are provided to facilitate understanding of the present invention. The following examples are provided to further understand the present invention, but the present invention is not limited by the examples.

[ Example ]

Example  1. Experimental instruments, instruments and materials

1-1. Experimental equipment

LC-MS / MS, which is an experimental apparatus used in the present invention, is a device (Liquid Chromatography coupled to Tandem Mass Spectrometry) in which a mass spectrometer of Table 1 is connected in series to a liquid chromatograph equipped with a column and HPLC shown in the following Table 1 .

[Table 1]

1-2. Experimental equipment

The experimental apparatus used in the present invention is shown in Table 2 below.

[Table 2]

1-3. reagent

The reagents used in the present invention are shown in Table 3 below.

[Table 3]

The structures of CP, PP, HexaP, HeptaP, UP, and coproporphyrin I- ¹⁵ N _{4 in} the above table are shown in FIG. 2 (A).

1-4. Mobile phase

Mobile phase A was prepared by adding 2 mL of formic acid to 998 mL of distilled water, and mobile phase B was prepared by adding 1 mL of formic acid to 999 mL of acetonitrile.

1-5. Washing solvent ( wash solvent )

1000 mL of methanol was mixed with 1000 mL of distilled water, and 2000 mL of 50% methanol was prepared as a washing solvent.

Example 2. LC-MS / MS Analysis Conditions and Numerical Handling

2-1. LC-MS / MS analysis conditions

The specific LC-MS / MS conditions used in the present invention are shown in Table 4 below.

[Table 4]

2-2. Handling of numerical values

The results obtained from LC-MS / MS were treated as shown in Table 5 below. Numbers are rounded to N (N + 1) digits.

[Table 5]

Example 3. Confirmation of sensitivity increase of esterified porphyrin on LC-MS / MS

3-1. Preparation of Control and Test Samples

(1) Preparation of intact porphyrin-free blood

100 mL of whole blood was prepared. 10 g of activated carbon was added to 100 mL of whole blood and mixed for 20 hours or more to remove porphyrin inherently contained in blood. The activated carbon was then removed from the blood using the centrifuge.

(2) Preparation of control sample

CP, PP, HexaP, HeptaP, and UP purchased from Frontier Scientific were added to the blood prepared in (1) above to prepare solutions each containing 10 nmol of each of the above compounds. The prepared solution was dropped onto a blood filter paper as shown in FIG. 1 and dried at room temperature and light-shielding conditions for 3 hours to prepare a dried blood spot sample. To the control sample, 200 μL of 0.5 M diluted formic acid distilled water was added to elute the compounds from the blood filter paper.

(3) Preparation of test sample

In the same manner as in the control sample, CP, PP, HexaP, HeptaP and UP purchased from Frontier Scientific Co. were added to the blood prepared in (1) above to prepare solutions each containing 10 nmol of each of the above compounds. The prepared solution was dropped on a blood filter paper as shown in FIG. 1 and dried at room temperature and light-shielding conditions for 3 hours to prepare a test sample (Dried Blood Spot sample).

To the test sample was added 500 μL of a solution containing 200 μL of 0.5 M diluted formic acid distilled water and a 9: 1 mixture of methanol (MeOH): sulfuric acid (H ₂ SO ₄ ), followed by vortexing for 50 minutes. Then, 1 mL of chloroform was added and the mixture was vortexed for 30 minutes and left for 30 minutes. After 30 minutes, the layer was separated, centrifuged at 13,000 rpm for 5 minutes, and 900 μL of the bottom layer was taken out and completely dried at 40 ° C. in a nitrogen evaporator. The dried sample was redissolved by adding 100 μL of acetonitrile.

3-2. Esterification of porphyrin pretreated with methanol and sulfuric acid

PP, HexaP, HeptaP, UP, and coproportin I- ¹⁵ N (v / v) in the case of pretreatment with a solution of methanol: sulfuric acid 9: ₄ was prepared in the same manner as in the reaction scheme below by using a mixture of Coproporphyrin tetramethyl ester (CPTE), Penta-carboxylic porphyrin pentamethyl ester (PPPE), Hexacarboxy porphyrin hexamethyl ester (Hexa-carboxylic porphyrin hexamethyl ester, hereinafter referred to as HexaPHE), hepta-carboxylic porphyrin heptamethyl ester (hereinafter referred to as HeptaPHE), Uroporphyrin octamethyl ester (hereinafter referred to as UPOE), coproporphyrin tetramethyl ester I- ¹⁵ N ₄ (Coproporphyrin I- ¹⁵ N ₄ tetramethyl ester).

[Reaction Scheme]

The structures of CPTE, PPPE, HexaPHE, HeptaPHE, UPOE and co-oporporphine tetramethyl ester I- ¹⁵ N ₄ are shown in FIG. 2 (B).

3-3. Perform LC-MS / MS and confirm increased sensitivity of esterified porphyrin

Take 5 μL of the control sample prepared in 3-1 above LC-MS / MS and analyzed. The analysis results are shown in Fig. 3 (A).

Further, in the test sample redissolved in acetonitrile prepared in 3-1 above, 5 μL was taken into LC-MS / MS and analyzed. The results of the analysis are shown in Fig. 3 (B).

On the graph of FIG. 3, the X axis represents time (minutes) and the Y axis represents concentration (cps). Each peak corresponds to CPTE, PPPE, HexaPHE, HeptaPHE and UPOE from the right. Since the mass of each compound increases at the time of esterification, the position where the peaks appear is shifted to the right as compared with FIG. 3 (A) in FIG. 3 (B).

In FIG. 3 (A), the CP shows a strength of 9600 cps, while in FIG. 3 (B), the CPTE shows an intensity of 26,000 cps. In the case of PPPE, HexaPHE, HeptaPHE and UPOE, the signal intensity was greatly increased as well. Therefore, when porphyrin was pretreated with a mixed solution of methanol and sulfuric acid, the sensitivity was greatly increased by LC-MS / MS.

Example 4. Evaluation of experiment

The calibration sample and the QC sample were prepared as described below to evaluate system suitability, specificity, carry-over, accuracy, and precision items to determine whether the experiment of Example 3 was properly performed.

4-1. Preparation of Standard Solution, Quality Control Solution and Internal Standard Solution

(1) Preparation of standard solution

0.75 mg of CP, 0.86 mg of PP, 0.91 mg of HexaP, 0.96 mg of HeptaP and 1.00 mg of UP were respectively weighed and dissolved by adding 100 mL of 6M formic acid (diluting solvent is distilled water) to obtain CP, PP, HexaP , HeptaP, and UP were prepared and stored frozen at 10,000 nmol / L.

Each of the above-mentioned stock solutions was serially diluted with 6M formic acid as shown in Table 6 to prepare a working solution.

[Table 6]

(2) Preparation of Quality Control Solution

The standard solution solutions of CP, PP, HexaP, HeptaP and UP prepared in (1) above were sequentially diluted with 6M formic acid as shown in Table 7 below to prepare QC solutions for QC solutions. The types of solutions (A), (E), and (F) taken in Table 7 are as shown in Table 6 above. In Table 7, QH denotes a high concentration QC solution (H: High), QM denotes a medium concentration QC solution (M: Medium), QL denotes a low concentration QC solution (L: Low), LLOQ denotes a minimum quantitation limit Lowest Limit of Quantification).

[Table 7]

(3) Preparation of internal standard solution

Copoloporphyrin I- ¹⁵ N ₄ 0.75 mg of sodium hydrogen sulphate was weighed and dissolved in 100 mL of 6M formic acid to prepare a stock solution of 10,000 nmol / L.

Before the experiment, the storage solution was diluted as shown in Table 8 to prepare a working solution.

[Table 8]

4-2. Preparation of sample

(1) Preparation of blanks

100 mL of whole blood was prepared. 10 g of activated carbon was added to 100 mL of whole blood and mixed for 20 hours or more to remove porphyrin inherently contained in blood. The activated carbon was then removed from the blood using the centrifuge.

(2) Preparation of calibration sample

A calibration sample was prepared by adding the standard solution working solution prepared in 4-1 (1) to the blank prepared in the above method (1) as shown in Table 9 below. The types of solutions taken from the following table are as shown in Table 6 above.

[Table 9]

Each of the prepared calibration curve solutions was dropped on blood filter paper as shown in FIG. 1 and dried at room temperature and light-shielding conditions for 3 hours to prepare a dried blood spot sample.

(3) Preparation of QC samples

Each QC solution prepared in the above 4-1 (2) was added to the blank prepared in the above method (1) as shown in Table 10 below to prepare a QC sample. The types of solutions taken from the following table are as shown in Table 7 above.

[Table 10]

Each QC solution thus prepared was dropped on blood filter paper as shown in FIG. 1, and dried for 3 hours at room temperature and light-shielding conditions to prepare a QC sample (Dried Blood Spot sample).

 4-3. Pretreatment of sample

In each of the calibration sample and the QC sample prepared in 4-2 above, coproporphyrin I- ¹⁵ N ₄ (diluted in a 6M formic acid solution at a concentration of 50 nmol / L), which is the internal standard solution working solution prepared in 4-1 (3) 10 μL was added. Then, elution and pretreatment were carried out by using 200 μL of diluted 0.5 M formic acid distilled water and a 9: 1 mixture of methanol (MeOH): sulfuric acid (H ₂ SO ₄ ) in the same procedure as in 3-1 Dried and redissolved in acetonitrile.

4-4. System suitability, specificity, carry-over, linearity and reproducibility evaluation

After pre-treatment prepared in 4-3 above, 5 μL of the supernatant of the redissolved solution in acetonitrile was injected into the LC-MS / MS in each case to evaluate each item.

(1) Evaluation of system suitability

The suitability of the present study equipment for securing objectivity to the experiment of Example 3 was confirmed by the following method. The QL samples were injected into LC-MS / MS in the S1 and QC samples in the calibration curve to ensure sensitivity. The criterion is that the precision (CV,%) of the peak area ratio for CPTE, PPTE, HexaPHE, HeptaPHE and UPOE is 10% or less in principle. However, %.

 The accuracy of CPTE was 2.23 ~ 9.30% in all batches. The precision of PPPE was 2.32 ~ 3.92%, the precision of HexaPHE was 3.17 ~ 8.94%, the precision of HeptaPHE was 3.14 ~ 8.67%, the precision of UPOE was 2.92 And 8.97%, respectively. The specific precision values in each case are shown in Table 11 below.

[Table 11]

(2) Evaluation of Specificity

The porphyrin-free whole blood, which corresponds to the blank sample, was interfered with the substance to be analyzed and confirmed by the following method whether or not there is a substance which interferes with selective and accurate analysis of the substance to be analyzed.

There are substances interfering with CPTE, PPTE, HexaPHE, HeptaPHE, and UPOE by injecting LLOQ (minimum quantitative limit) samples in QC samples prepared by using 6 whole blood of different origins into LC-MS / MS Respectively. The criterion was that there was no interfering material if there was no material showing more than 20% of the peak area of the daily LLOQ sample and more than 5% of the internal standard.

The results are shown in Figs. 4 to 8 (there is a difference in the position where the peaks appear within the range that can be understood by those skilled in the art with Fig. 3 (B) due to LC-MS / MS characteristics). Each diagram (A) shows the result of mass analysis of the blank sample, and (B) shows the result of analysis of each compound. In FIG. 4, it can be seen that the blank sample does not contain a substance having a peak appearing at 8.59 minutes corresponding to the peak of CPTE. In FIG. 5, it can be seen that the blank sample does not contain a substance having a peak at 7.64 min corresponding to the peak of PPPE. In FIG. 6, it can be seen that the blank material does not contain a substance having a peak appearing at 6.88 minutes corresponding to HexaPHE. In FIG. 7, it can be seen that the blank material does not contain a substance having a peak appearing at 6.24 min corresponding to HeptaPHE. In FIG. 8, it can be seen that the blank material does not contain a substance having a peak appearing at 5.77 minutes corresponding to UPOE. In other words, it was confirmed that there was no substance interfering with CPTE, PPTE, HexaPHE, HeptaPHE and UPOE in the porphyrin-removed whole blood used in this experiment.

(3) carry-over evaluation

When the sample was injected into the LC-MS / MS and the next sample was injected, it was confirmed by the following method whether it was affected by the sample injected before. First, a blank sample was injected into LC-MS / MS. A sample (Upper Limit Of Quantification, ULOQ) of 50 nmol / L, which is the highest concentration sample in the calibration curve, was injected into LC-MS / MS, The results of this study are as follows.

As a result, no carry-over effect was observed.

(4) Evaluation of Linearity

Using the correlation coefficient and accuracy obtained from the calibration curve samples, it was confirmed whether the experimental method can obtain a linear measurement of the concentration of the substance to be analyzed within a certain range.

CPTE, PPTE, HexaPHE, HeptaPHE, and UPOE were injected into LC-MS / MS, and calibration curves were obtained based on the results. The acceptance criterion is that the deviation from the theoretical concentration should be within 20% of LOQ (minimum quantitative limit) and the deviation from the theoretical concentration should be less than 15% and the correlation coefficient should be at least 0.98 at concentrations other than LOQ.

In the range of 0.2 to 50 nmol / L, the correlation coefficients (r) were 0.9998 to 0.9999 for CPTE, 0.9815 to 0.9999 for PPPE, 0.9958 to 0.9999 for HexaPHE, 0.9938 to 0.9986 for HeptaPHE and 0.9980 to 0.9990 for UPOE, And accuracy were all satisfied. Specific values are shown in Tables 12 to 16 below.

[Table 12]

[Table 13]

[Table 14]

[Table 15]

[Table 16]

(5) Reproducibility (accuracy and precision) evaluation

Accuracy was calculated as the percentage of the ratio obtained by dividing the average concentration obtained by substituting QC samples into LC-MS / MS with calibration curve and dividing by the concentration of the actual QC sample. Intra-day accuracy was calculated five times a day in each of the QH, QM, QL, and LLOQ samples, and the inter-day accuracy was calculated by repeating the experiment for 5 days.

The acceptance criteria should be within ± 15% of the theoretical value except for the lowest quantitative limit in the single component analysis, and the average value should not exceed ± 20% of the theoretical value at the lowest quantitative limit, Of the theoretical concentration at all concentrations.

Precision is the average value of the peak area ratio of CPTE, PPTE, HexaPHE, HeptaPHE and UPOE obtained by injecting QC samples into LC-MS / MS and internal standard COPORPORPHYLPETRIN Tetramethylester I- ¹⁵ N ₄ (%) Of the divided ratio. QH, QM, QL, and LLOQ samples were performed 5 times a day to calculate the intra-day precision and the daily precision was calculated by repeating the experiment for 5 days.

The tolerance criteria is that the CV should be within 15% of the precision measured for each concentration in single component analysis, and the CV should not exceed 20% at the lowest quantitative limit. However, The CV was within 20% at all concentrations.

The intra-day reproducibility for accuracy and precision was as follows.

The precision of CPTE was 4.35%, 4.76%, 9.20% and 6.48% in LLOQ, QL, QM and QH, respectively. Accuracy was 114.63%, 109.10%, 88.06% and 106.51% in LLOQ, QL, Respectively. Specific values are shown in Table 17 below.

[Table 17]

The precision of PPPE was 4.14%, 7.58%, 5.93%, and 5.39% in LLOQ, QL, QM and QH respectively and the accuracy was 108.69%, 103.64%, 92.87% and 93.32% in LLOQ, QL, Respectively. Specific values are shown in Table 18 below.

[Table 18]

The precision of HexaPHE was 11.53%, 4.88%, 2.63% and 2.28% in LLOQ, QL, QM and QH respectively and the accuracy was 94.13%, 86.06%, 86.74% and 85.20% in LLOQ, QL, Respectively. Specific values are shown in Table 19 below.

[Table 19]

The precision of HeptaPHE was 17.07%, 7.57%, 14.27% and 13.15% in LLOQ, QL, QM and QH, respectively, and accuracy was 104.07%, 101.50%, 93.47% and 103.24% in LLOQ, QL, Respectively. Specific values are shown in Table 20 below.

[Table 20]

The accuracy of UPOE was 4.35%, 4.76%, 9.20% and 6.48% in LLOQ, QL, QM and QH, respectively. Accuracy was 114.63%, 109.10%, 88.06% and 106.51% in LLOQ, QL, Respectively. Specific values are shown in Table 21 below.

[Table 21]

The daily reproducibility for accuracy and precision was as follows.

The accuracy of CPTE was 4.80%, 12.11%, 12.71% and 7.14% in LLOQ, QL, QM and QH, and the accuracy was 114.72%, 103.08%, 97.49% and 109.05% in LLOQ, QL, QM and QH . Specific values are shown in Table 22 below.

[Table 22]

The precision of PPPE was 3.99%, 10.61%, 6.46% and 8.85% in LLOQ, QL, QM and QH, and the accuracy was 111.49%, 102.53%, 101.14% and 102.60% in LLOQ, QL, QM and QH . Specific values are shown in Table 23 below.

[Table 23]

The precision of HexaPHE was 8.30%, 6.91%, 10.91% and 12.62% in LLOQ, QL, QM and QH, and accuracy was 99.32%, 94.33%, 100.69% and 103.69% in LLOQ, QL, QM and QH . Specific values are shown in Table 24 below.

[Table 24]

The precision of HeptaPHE was 9.11%, 12.37%, 13.37% and 10.55% in LLOQ, QL, QM and QH, and the accuracy was 106.66%, 104.24%, 100.13% and 104.72% in LLOQ, QL, QM and QH . Specific values are shown in Table 25 below.

[Table 25]

The accuracy of UPOE was 12.90%, 13.09%, 9.87% and 10.08% in LLOQ, QL, QM and QH, and the accuracy was 105.35%, 105.42%, 101.94% and 101.99% in LLOQ, QL, QM and QH . Specific values are shown in Table 26 below.

[Table 26]

Claims (9)

1) collecting a biological sample from a subject;
2) pre-treating the biological sample with a mixed solution of methanol and strong acid; And
3) A method for analyzing porphyrin in a biological sample, comprising the step of analyzing the pretreated biological sample by LC-MS / MS. The method according to claim 1, wherein the pretreatment in step (2) comprises contacting or mixing a mixed solution of a biological sample and methanol and a strong acid. The method according to claim 1, wherein the biological sample in step (1) is blood. The process according to claim 1, wherein the strong acid in step (2) is sulfuric acid, hydrochloric acid or nitric acid. The method according to claim 1, wherein the mixed solution of methanol and strong acid in step 2) is mixed in a volume ratio of methanol: strong acid of 9: 1 to 8: 2. 2. The method of claim 1, wherein the step 1) comprises the following steps.
a) collecting a biological sample from a subject;
b) dropping the biological sample on a blood filter paper; and
c) eluting the biological sample from the blood filter paper. The method according to claim 6, wherein the elution of the biological sample in step c) is carried out by contacting formic acid with a blood filter paper from which the biological sample has been dropped. 2. The method of claim 1, wherein the subject is a human. The method of claim 1, wherein the porphyrin is selected from the group consisting of Coproporphyrin, Pentacarboxylporphyrin, Hexacarboxylporphyrin, Heptacarboxylporphyrin, and Uroporphyrin. Way.

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