WO2015142130A1

WO2015142130A1 - Method for detecting porphyrin in a biological sample by using lc-ms/ms

Info

Publication number: WO2015142130A1
Application number: PCT/KR2015/002779
Authority: WO
Inventors: Young Shin Lee; Seung Woo Kang; Yeoun Hur; Sang Beom Han; Ju Hee Jung; Kwang Jin Kim
Original assignee: Inernational Scientific Standards, Inc.
Priority date: 2014-03-21
Filing date: 2015-03-20
Publication date: 2015-09-24
Also published as: KR20150109995A

Abstract

Provided herein is a method for detecting porphyrin using LC-MS/MS by pretreating a biological sample. This method is very effective in detecting a small amount of porphyrin. Accordingly, it is possible to detect porphyrin from biological samples which contain a small amount of porphyrin such as blood. Furthermore, even in the case where the number of times obtaining biological samples and the amount of obtainable biological samples are very limited, such as newborn infants, a precise detection of porphyrin is possible. Hence, this method can be utilized for early diagnosis of porphyrin-related diseases.

Description

METHOD FOR DETECTING PORPHYRIN IN A BIOLOGICAL SAMPLE BY USING LC-MS/MS

The present invention relates to a method for detecting porphyrin by using LC-MS/MS (Liquid Chromatography coupled to Tandem Mass Spectrometry).

Together with iron, porphyrin makes up heme, which is a component of hemoglobin. Porphyrin is also a component of cytochrome, which plays an important role in oxidation and reduction in vivo. Further, porphyrin is a component of chlorophyll, which is involved in photosynthesis.

Porphyrin is related to various diseases. A typical example of porphyrin-related disease is porphyria, which is caused by accumulation of porphyrin and iron in internal organs caused by the lack of enzymes involved in the synthesis of heme due to inherited or acquired genetic defects. The common symptoms of Porphyria are neurovisceral or cutaneous symptoms. Neurovisceral symptoms appear after puberty and are more common in women, and include acute seizures, abdominal pain, and damaged sensorimotor nervous systems. Cutaneous symptoms, which appear after 30s in most cases, include excessive peeling, blisters and scars after being exposed to sunlight. Hepatopathy associated with the accumulation of porphyrin may appear together.

Methods to treat porphyria have not yet been developed. However, reducing exposure to sun and drugs, alcohol, female hormone, etc., or taking measures to prevent porphyrin from being accumulated in liver by periodic bloodletting early in the stage of porphyria is known to alleviate the symptoms. Mortality in patients who were not diagnosed early is reported to range from 10 ~ 40%.

Recently, porphyrin is receiving attention as a biomarker of autism or autistic spectrum disorder. There are reports that the concentration of porphyrin such as coproporphyrin, hexacarboxylporphyrin and pentacarboxylporphyrin is high in the urine of children with autism. Further, there are many papers reporting that excessive amount of porphyrin causes heavy metal such as mercury to remain longer in vivo and triggers autism in result. There are no methods available to treat autism now. However, Nafta et al.(2006) reported that chelators such as DMSA or ALA improved the symptoms of autism.

Although early diagnosis of porphyrin-related diseases by detecting and analyzing porphyrin is important, there is a limit to the tests for porphyrin to date. Porphyrin is excreted in urine, feces and bile, and is particularly released in a large amount in the urine. Porphyrin is also present in blood, but its amount is too small to detect in tests. Tests for porphyrin are usually performed by using LC-MS/MS. However, it is hard to detect a small amount of porphyrin with conventional methods.

The purpose of the present invention is to provide a method for detecting porphyrin in a biological sample using LC-MS/MS, wherein the method includes pretreating the biological sample with a mixed solution of methanol and strong acid.

The present invention provides a method for detecting porphyrin in a biological sample, comprising: 1) obtaining a biological sample from a subject; 2) pretreating the biological sample with a mixed solution of methanol and strong acid; and 3) analyzing the pretreated biological sample by using LC-MS/MS.

In the method for the present invention, the subject may be a human.

In the method for the present invention, the pre-treatment of 2) may be performed by bringing the biological sample into contact with the mixed solution of methanol and strong acid or mixing the biological sample with the mixed solution of methanol and strong acid.

As used herein, the term “porphyrin” commonly denotes compounds having the following basic structure and derivatives thereof.

[Structural Formula]

The form of porphyrin may vary. Protoporphyrin, harderoporphyrin, coproporphyrin, pentacarboxylporphyrin, hexacarboxylporphyrin, heptacarboxylporphyrin, and uroporphyrin, etc. are generally present in blood, and excreted in urine and feces. They are all referred to as porphyrin. Furthermore, there are type I, II, and III for each porphyrin, and they are all referred to as porphyrin.

Many kinds of porphyrin include one or more carboxyl groups. For example, protoporphyrin has two carboxyl groups, harderoporphyrin has three carboxyl groups, coproporphyrin has four carboxyl groups, pentacarboxylporphyrin has five carboxyl groups, hexacarboxylporphyrin has six carboxyl groups, heptacarboxylporphyrin has seven carboxyl groups, and uroporphyrin has eight carboxyl groups. As a porphyrin has more carboxyl groups, the porphyrin is more soluble to water and more likely to be excreted in urine. Porphyrins with 4 carboxyl groups tend to be excreted in urine or feces, and harderoporphyrin or protoporphyrin tend to be excreted in feces.

LC-MS/MS (Liquid Chromatography coupled to Tandem Mass Spectrometry) is composed of more than one mass spectrometers arranged in a row and connected to a liquid chromatography instrument consisting of columns and HPLC (High Performance Liquid Chromatography). In LC-MS, a sample is injected into the column by using HPLC and the components contained in the sample are separated. Then the components enter the Mass spectrometer and are ionized, and show signal peaks at different times, from which the corresponding materials may be identified. LC-MS/MS, which has more than one (typically two) mass spectrometers, enables more accurate analysis of materials. Since LC-MS/MS is capable of separating materials in samples and identifying the materials, it is widely used in analysis of a variety of materials.

However, it is very difficult to analyze compounds which have carboxyl groups, such as porphyrin, by using LC-MS/MS. Since carboxyl groups interfere the ionization of porphyrin in mass spectrometer, the sensitivity of the instrument is decreased and the precision of the analysis is impaired in result. The method of the present invention includes pretreating the obtained biological sample with the mixed solution of methanol and strong acid, and thereby porphyrin is esterified so that the carboxyl group is substituted with a methyl ester group and the produced water is removed, ultimately enabling precise detection of porphyrin.

Since the method for the present invention is able to detect a small amount of porphyrin, it is more useful when the amount of biological sample to be obtained is small. One example where only a small amount of sample can be obtained is the case of newborn infants. Since the newborn infants are small in size and very sensitive to stimulation, the number of times obtaining biological samples and the amount of obtainable biological samples are limited. When using the method of the present invention, it is possible to detect porphyrin in a droplet of blood, which is about 3 μL. Hence, in the present invention, the subjects include but not limited to a baby within one month of birth, more preferably seven days of birth, and much more preferably 72 hours of birth.

One example the method of the present invention can be utilized is the neonatal screening tests. Some of congenital metabolic disorders may cause irreparable damage upon late diagnosis but such damage may be prevented by treating in early stage. Hence, the Korean Government supports neonatal screening tests for six kinds of congenital metabolic disorders with high incidence rate, and most parents additionally perform tests for the other dozens of diseases. These tests are carried out in a manner that the blood is drawn from the heel of a 3 ~ 7 day-old baby, dropped on a blood filter paper, dried, and analyzed in lab. As such, the blood filter paper contains only about 3 μL of blood. Since the method of the present invention enables the detection of porphyrin using blood of the blood filter paper, there is no need to obtain more biological sample from a subject to detect porphyrin.

In the method of the present invention, the porphyrins include but not limited to porphyrins with four or more carboxyl groups, such as coproporphyrin, pentacarboxylporphyrin, hexacarboxylporphyrin, heptacarboxylporphyrin, or uroporphyrin.

Furthermore, the method of the present invention is effective in detecting porphyrin from a biological sample containing a small amount of porphyrin. Hence, the biological samples in 1) include but not limited to blood, for example, whole blood, plasma and serum. The biological samples in 1) include but not limited to urine, feces, and bile.

In the method of the present invention, the strong acids in 2) include but not limited to sulfuric acid, hydrochloric acid, and nitric acid, preferably sulfuric acid.

In the method of the present invention, the mixing ratio of methanol and strong acid in 2) is 9:1 ~ 8:2 (v/v), and preferably 9:1. However, the mixing ratio is not limited thereto.

In the method of the present invention, 1) may include:

a) obtaining 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 an embodiment of the present invention, eluting the biological sample in c) may be conducted by bringing formic acid into contact with the blood filter paper on which the biological sample was dropped. The use of formic acid enables effective elution of the blood contained in the porous blood filter paper. Preferably 0.5 M formic acid in distilled water can be used. However, the concentration of the solution which can be used in the method herein is not limited thereto.

By using the present invention, a small amount of porphyrin can be detected effectively. Accordingly, it is possible to detect porphyrin from biological samples containing a small amount of porphyrin such as blood. Even in the case where the number of times obtaining biological samples and the amount of obtainable biological samples are very limited, such as newborn infants, a precise detection of porphyrin is possible. Therefore, the method of the present invention can be utilized for early diagnosis of porphyrin-related diseases.

FIG. 1 is a schematic illustration of a blood filter paper;

FIG. 2A illustrates the structures of coproporphyrin (CP), pentacarboxylporphyrin (PP), hexacarboxylporphyrin (HexaP), heptacarboxylporphyrin (HeptaP), uroporphyrin (UP), and coproporphyrin I-¹⁵N₄, and FIG. 2B illustrates the structures of their esterified forms;

FIG. 3A illustrates the results of mass analysis of CP, PP, HexaP, HeptaP, and UP, and FIG.3B illustrates the results of mass analysis of their esterified forms;

FIG. 4A illustrates the results of analysis of LC-MS/MS of a blank, and FIG. 4B illustrates the results of analysis of LC-MS/MS of a 50 nmol/L CPTE calibration solution;

FIG. 5A illustrates the results of analysis of LC-MS/MS of a blank, and FIG. 5B illustrates the results of analysis of LC-MS/MS of a 50 nmol/L PPPE calibration solution;

FIG. 6A illustrates the results of analysis of LC-MS/MS of a blank, and FIG. 6B illustrates the results of analysis of LC-MS/MS of a 50 nmol/L HexaPHE calibration solution;

FIG. 7A illustrates the results of analysis of LC-MS/MS of a blank, and FIG. 7B illustrates the results of analysis of LC-MS/MS of a 50 nmol/L HeptaPHE calibration solution; and

FIG. 8A illustrates the results of analysis of LC-MS/MS of a blank, and FIG. 8B illustrates the results of analysis of LC-MS/MS of a 50 nmol/L UPOE calibration solution.

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[Examples]

Example 1. Testing instruments, testing devices, and materials

1-1. Testing instrument

LC-MS/MS used in the Examples herein includes the mass spectrometers and columns listed in the following Table 1.

[Table 1]

1-2. Testing devices

The testing devices used in the Examples herein are as in the Table 2 below.

[Table 2]

1-3. Reagents

The reagents used in the Examples herein are as in the Table 3 below.

[Table 3]

The structures of CP, PP, HexaP, HeptaP, UP, and coproporphyrin I-¹⁵N₄ in the above Table 3 are illustrated in FIG. 2A.

1-4. Mobile phase

To 998 mL of distilled water (DW), 2 mL of formic acid was added to prepare a mobile phase A, and to 999 mL of acetonitrile, 1 mL of formic acid was added to prepare a mobile phase B.

1-5. Wash solvent

To 1000 mL of methanol, 1000 mL of distilled water was added to prepare 2000 mL of 50% methanol, which used as a wash solvent.

Example 2. LC-MS/MS analysis conditions and numerical treatment of values

2-1. LC-MS/MS analysis conditions

The specific conditions for LC-MS/MS used in the Examples herein are described in Table 4 below.

[Table 4]

2-2. The numerical treatment of values

The numerical values obtained from the result of LC-MS/MS were treated as described in Table 5 below. The numerical values are shown as N decimal points by rounding off the (N+1) decimal points.

[Table 5]

Example 3. The increase in sensitivity of esterified porphyrin in LC-MS/MS

3-1. Preparation of control and testing samples

(1) Preparation of intrinsic porphyrin-free blood

100 mL of whole blood was prepared. 10 g of activated carbon was added to the 100 mL of the whole blood, and mixed for at least 20 hours to remove porphyrin intrinsically contained in the blood. Then the activated carbon was removed from the blood by using a centrifuge.

(2) Preparation of control samples

CP, PP, HexaP, HeptaP, and UP, which were purchased from Frontier Scientific, were added to the blood prepared in (1), and solutions containing the above compounds at a concentration of 10 nmol/L respectively were prepared. Each of the prepared solutions was dropped on a blood filter paper schematically illustrated in FIG. 1, and dried at room temperature for at least 3 hours out of sunlight to prepare control samples (dried blood spot samples). Then 200 μL of 0.5 M formic acid in DW was added to the control sample to elute the compounds from the blood filter paper.

(3) Preparation of testing samples

Like control samples, CP, PP, HexaP, HeptaP, and UP, which were purchased from Frontier Scientific, were added to the blood prepared in (1), and solutions containing the above compounds at a concentration of 10 nmol/L respectively were prepared. Each of the prepared solutions was dropped on a blood filter paper schematically illustrated in FIG. 1, and dried at room temperature for at least 3 hours out of sunlight to prepare testing samples (dried blood spot samples).

Then 200 μL of 0.5 M formic acid in DW, and 500 μL of 9:1(v/v) solution of methanol (MeOH) and sulfuric acid (H₂SO₄) were added to the test samples, and then the samples were vortex-mixed for 50 min. Subsequently, to the resulting mixture, 1 mL of chloroform was added, and the mixture was vortex-mixed for 30 min, and allowed to stand for 30 min. After 30 min, layer separation was confirmed, centrifugation was performed at 13,000 rpm for 5 min, and 900 μL of the lower layer was completely dried at 40 ℃ using a nitrogen evaporator. To the dried samples, 100 μL of acetonitrile was added to re-dissolve the samples.

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

When pre-treatment was carried out with a 9:1 (v/v) solution of methanol and sulfuric acid as in 3-1(3) through a separate assay, CP, PP, HexaP, HeptaP, UP and coproporphyrin I-¹⁵N₄ were esterified via the reaction mechanism below to coproporphyrin tetramethyl ester (CPTE), pentacarboxylporphyrin pentamethyl ester (PPPE), hexacarboxylporphyrin hexamethyl ester (HexaPHE), heptacarboxylporphyrin heptamethyl ester (HeptaPHE), uroporphyrin octamethyl ester (UPOE), and coproporphyrin I-¹⁵N₄ tetramethyl ester, respectively.

[Scheme]

The structures of CPTE, PPPE, HexaPHE, HeptaPHE, UPOE and coproporphyrin tetramethyl ester I-¹⁵N₄ are illustrated in FIG. 2B.

3-3. Increased sensitivity of esterified porphyrin in LC-MS/MS

5 μL of each of the control samples prepared in 3-1 was injected into LC-MS/MS to be analyzed. The analysis results are shown in FIG. 3A.

Also, 5 μL of the upper layer of each of the testing samples re-dissolved in acetonitrile prepared in 3-1 was injected into LC-MS/MS to be analyzed. The analysis results are shown in FIG. 3B.

On the graphs of FIGS. 3A and 3B, the X axis represents time (min) and the Y axis represents the concentration (cps). Each peak corresponds to CPTE, PPPE, HexaPHE, HeptaPHE, and UPOE from the right. Since the mass of each compound is increased upon esterification, the peaks of FIG. 3B were shifted rightward compared to FIG. 3A.

As illustrated in FIG. 3A, while CP showed an intensity of 9600 cps, the intensity of CPTE showed an intensity of 26,000 cps. As for PPPE, HexaPHE, HeptaPHE, and UPOE, such increases in signal intensities were also observed. Thus, it was understood that the pre-treatment of porphyrin with the mixed solution of methanol and sulfuric acid significantly increased the sensitivity in LC-MS/MS.

Example 4. Evaluation of the tests

Calibration standards and quality control (QC) samples were prepared as follows, and measured to check system suitability, specificity, carry-over, accuracy and precision, thereby evaluating whether the tests of Example 3 were appropriately performed.

4-1. Preparation of standard solution, QC 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 weighed, dissolved in 100 mL of 6 M formic acid in DW, to prepare 10,000 nmol/L stock solutions for CP, PP, HexaP, HeptaP, and UP, respectively. The stock solutions were then frozen and stored.

Before the tests, each stock solution was subjected to serial dilution with 6 M formic acid as shown in Table 6 below, to prepare working solutions.

[Table 6]

(2) Preparation of QC solution

The working solutions for CP, PP, HexaP, HeptaP, and UP prepared in (1) were subjected to serial dilution with 6 M formic acid as shown in Table 7 below, to prepare QC solutions. The kinds of solutions taken in Table 7 below ((A), (E), (F)) are as represented in Table 6. In Table 7 below, QH denotes high-concentration QC solution (H: High), QM denotes medium-concentration QC solution (M: Medium), QL denotes low-concentration QC solution (L: Low), and LLOQ denotes a QC solution having a concentration corresponding to the lowest limit of quantification.

[Table 7]

(3) Preparation of internal standard solution

0.75 mg of coproporphyrin I-¹⁵N₄ sodium bisulfate was weighed, and dissolved in 100 mL of 6 M formic acid, to prepare 10,000 nmol/L stock solution, which was then frozen and stored.

Before the tests, the stock solution was diluted as shown in Table 8 below, to prepare a working solution.

[Table 8]

4-2. Preparation of samples

(1) Preparation of a blank

(2) Preparation of calibration standard

By adding the working solutions prepared in 4-1(1) as shown in Table 9 below to the blank prepared in (1), the calibration standards were prepared. The kinds of solutions taken in the following table are as represented in Table 6.

[Table 9]

Each of the prepared calibration solutions was dropped on the blood filter paper as schematically illustrated in FIG. 1, and dried at room temperature for at least 3 hours out of sunlight to prepare calibration standards (dried blood spot samples).

(3) Preparation of QC samples

QC solutions prepared in 4-1(2) were added to the blank prepared in (1) to prepare QC samples, as shown in Table 10 below. The kinds of solutions taken in the following Table 10 are as represented in Table 7.

[Table 10]

Each of the prepared QC solutions was dropped on the blood filter paper as schematically illustrated in FIG. 1, and dried at room temperature for at least 3 hours out of sunlight, to prepare QC samples (dried blood spot samples).

4-3. Pre-treatment of samples

To each of the calibration standards and the QC samples prepared in 4-2, 10 μL of coproporphyrin I-¹⁵N₄ (50 nmol/L 6 M formic acid solution), the internal working solution prepared in 4-1(3), was added. Then, elution and pre-treatment were performed in the same manner as in 3-1(3) using 200 μL of a 0.5 M formic acid in DW and a 9:1 (v/v) solution of methanol (MeOH) and sulfuric acid (H₂SO₄), followed by drying and re-dissolution in acetonitrile.

4-4. Evaluation of system suitability, specificity, carry-over, linearity, and reproducibility

5 μL of the upper layer of the pretreated solution prepared in 4-3 was injected into LC-MS/MS to evaluate the following items.

(1) Evaluation of system suitability

The suitability of the present tester to ensure objectivity of the tests of Example 3 was evaluated as below. S1, among the calibration standards, and QL, among the QC samples, were injected into LC-MS/MS to ensure sensitivity. As an acceptance criterion for system suitability, the precision (CV, %) of the peak area ratio of each of CPTE, PPPE, HexaPHE, HeptaPHE, and UPOE relative to the internal standard is 10% or less in principle, but was set to 15% in the present tests because five kinds of materials were analyzed simultaneously.

In all batches, the precision of CPTE was 2.23 ~ 9.30%, 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%, and the precision of UPOE was 2.92 ~ 8.97%, all of which satisfied the acceptance criterion. Specific numerical values for precision are shown in Table 11 below.

[Table 11]

(2) Evaluation of specificity

To determine whether materials which interfere the subject material to be analyzed and hinder the selective and accurate analysis of the subject material are present in the whole blood from which porphyrins are removed, the following evaluation was conducted.

Blanks prepared by using six kinds of whole blood from different origins, and the LLOQ sample, among the QC samples, were injected into LC-MS/MS, and whether materials that interferes CPTE, PPPE, HexaPHE, HeptaPHE, and UPOE were present in the sample was determined. As an acceptance criterion for specificity, when there was no material that exceeds 20% of the peak area of the LLOQ sample for intra-day assays and exceeds 5% for the internal standard, the sample was determined to be free of any material interfering the subject material.

The results are shown in FIGS. 4 to 8 (for LC-MS/MS, there are slight differences in the positions of peaks from those in FIG. 3B. Such differences are within the range understood by one skilled in the art). FIGS. 4A to 8A show the results of mass analysis of the blanks, and FIGS. 4B to 8B show the results of analysis of each compound. In FIGS. 4A and 4B, it can be understood that the blank does not include any material showing a peak at 8.59 min, which corresponds to the peak of CPTE. In FIGS. 5A and 5B, it can be understood that the blank does not include any material showing a peak at 7.64 min, which corresponds to the peak of PPPE. In FIGS. 6A and 6B, it can be understood that the blank does not include any material showing a peak at 6.88 min, which corresponds to the peak of HexaPHE. In FIGS. 7A and 7B, it can be understood that the blank does not include any material showing a peak at 6.24 min, which corresponds to the peak of HeptaPHE. In FIGS. 8A and 8B, it can be understood that the blank does not include any material showing a peak at 5.77 min, which corresponds to the peak of UPOE. Accordingly, the porphyrin-free whole blood used in the tests did not include any material that interfere CPTE, PPPE, HexaPHE, HeptaPHE, and UPOE.

(3) Evaluation of carry-over

When a series of samples were sequentially injected into LC-MS/MS, whether a sample was influenced by the previous sample was evaluated as below. At first, a blank was injected into LC-MS/MS, and 50 nmol/L ULOQ sample, which has the highest concentration among calibration standards, was injected into LC-MS/MS, and then the blank was injected into the instrument again. As such, whether there is a carry-over effect caused by the previously injected ULOQ sample was determined.

Consequently, no carry-over effect was observed.

(4) Evaluation of linearity

By using the accuracy and the correlation coefficient obtained from the calibration standards, whether the measured values could be linear with respect to the concentration of the analyte material within a predetermined range by the present testing method was evaluated.

Based on the results of analysis of the calibration standards for CPTE, PPPE, HexaPHE, HeptaPHE, and UPOE using LC-MS/MS, a calibration curve was drawn, from which correlation coefficients were determined. An acceptance criterion is as follows: a deviation from the nominal concentration at LOQ has to be within 20% and a deviation from the nominal concentration at a concentration other than LOQ has to be within 15%, where r (correlation coefficient) has to be 0.98 or more.

The correlation coefficient (r) in the concentration range of 0.2 ~ 50 nmol/L is 0.9982 ~ 0.9999 for CPTE, 0.9815 ~ 0.9999 for PPPE, 0.9958 ~ 0.9999 for HexaPHE, 0.9938 ~ 0.9986 for HeptaPHE, and 0.9980 ~ 0.9990 for UPOE, all of which were equal to or higher than the acceptance criterion, and the accuracy results satisfied the acceptance criterion. The specific numerical values thereof are provided in Tables 12 to 16 below.

[Table 12]

[Table 13]

[Table 14]

[Table 15]

[Table 16]

(5) Evaluation of reproducibility (Accuracy and Precision)

Accuracy was expressed as a percentage (%) of the value obtained in such a manner that the numerical values obtained from injecting QC samples into LC-MS/MS were substituted into the calibration curves to give the mean concentration that was then divided by the concentration of the actual QC samples as known. Five tests were performed per day using QH, QM, QL, and LLOQ samples to calculate intra-day accuracy, and the tests were repeated for five days to calculate inter-day accuracy.

An acceptance criterion for accuracy is as follows: upon analysis of single component, the mean value has to be within ±15% of the nominal value except for LLOQ, and at LLOQ, the mean value does not exceed ±20% of the nominal value. However, since five kinds of materials were simultaneously analyzed in the present tests, the criterion was set within 20% of the nominal concentration in all concentrations.

The precision was expressed as a percentage (%) of the value obtained in such a manner that the numerical values resulting from injecting the QC samples in LC-MS/MS were divided by the mean value of the peak area ratios of CPTE, PPPE, HexaPHE, HeptaPHE, and UPOE and coproporphyrin tetramethyl ester I-¹⁵N₄ as the internal standard. Five tests were performed per day using QH, QM, QL, and LLOQ samples to calculate intra-day precision, and the tests were repeated for five days to calculate inter-day precision.

An acceptance criterion for precision is as follows: upon analysis of single component, CV has to be within 15% at each concentration, and CV should not exceed 20% at LLOQ. However, since five kinds of materials were simultaneously analyzed in the present tests, CV was set within 20% in all concentrations.

The intra-day reproducibility results for accuracy and precision are as below.

The precision of CPTE was 4.35%, 4.76%, 9.20% and 6.48% at LLOQ, QL, QM and QH, respectively. The accuracy of CPTE was 114.63%, 109.10%, 88.06% and 106.51% at LLOQ, QL, QM and QH, respectively. The specific numerical values thereof are provided in Table 17 below.

[Table 17]

The precision of PPPE was 4.14%, 7.58%, 5.93% and 5.39% at LLOQ, QL, QM and QH, respectively, and the accuracy of PPPE was 108.69%, 103.64%, 92.87% and 93.32% at LLOQ, QL, QM and QH, respectively. The specific numerical values thereof are provided in Table 18 below.

[Table 18]

The precision of HexaPHE was 11.53%, 4.88%, 2.63% and 2.28% at LLOQ, QL, QM and QH, respectively, and the accuracy of HexaPHE was 94.13%, 86.06%, 86.74% and 85.20% at LLOQ, QL, QM and QH, respectively. The specific numerical values thereof are provided in Table 19 below.

[Table 19]

The precision of HeptaPHE was 17.79%, 7.57%, 14.27% and 13.15% at LLOQ, QL, QM and QH, respectively, and the accuracy of HeptaPHE was 104.07%, 101.50%, 93.47% and 103.24% at LLOQ, QL, QM and QH, respectively. The specific numerical values thereof are provided in Table 20 below.

[Table 20]

The precision of UPOE was 4.35%, 4.76%, 9.20% and 6.48% at LLOQ, QL, QM and QH, respectively, and the accuracy of UPOE was 114.63%, 109.10%, 88.06% and 106.51% at LLOQ, QL, QM and QH, respectively. The specific numerical values thereof are provided in Table 21 below.

[Table 21]

The inter-day reproducibility results for accuracy and precision are shown below.

The precision of CPTE was 4.80%, 12.11%, 12.71% and 7.14% at LLOQ, QL, QM and QH, respectively. The accuracy of CPTE was 114.72%, 103.08%, 97.49% and 109.05% at LLOQ, QL, QM and QH, respectively. The specific numerical values thereof are provided in Table 22 below.

[Table 22]

The precision of PPPE was 3.99%, 10.61%, 6.46% and 8.85% at LLOQ, QL, QM and QH, respectively. The accuracy of PPPE was 111.49%, 102.53%, 101.14% and 102.60% at LLOQ, QL, QM and QH, respectively. The specific numerical values thereof are provided in Table 23 below.

[Table 23]

The precision of HexaPHE was 8.30%, 6.91%, 10.91% and 12.62% at LLOQ, QL, QM and QH, respectively. The accuracy of HexaPHE was 99.32%, 94.33%, 100.69% and 103.69% at LLOQ, QL, QM and QH, respectively. The specific numerical values thereof are provided in Table 24 below.

[Table 24]

The precision of HeptaPHE was 9.11%, 12.37%, 13.37% and 10.55% at LLOQ, QL, QM and QH, respectively. The accuracy of HeptaPHE was 106.66%, 104.24%, 100.13% and 104.72% at LLOQ, QL, QM and QH, respectively. The specific numerical values thereof are provided in Table 25 below.

[Table 25]

The precision of UPOE was 12.90%, 13.09%, 9.87% and 10.08% at LLOQ, QL, QM and QH, respectively. The accuracy of HeptaPHE was 105.35%, 105.42%, 101.94% and 101.99% at LLOQ, QL, QM and QH, respectively. The specific numerical values thereof are provided in Table 26 below.

[Table 26]

Claims

A method for detecting porphyrin in a biological sample, comprising:

1) obtaining a biological sample from a subject;

2) pretreating the biological sample with a mixed solution of methanol and strong acid; and

3) analyzing the pretreated biological sample by using LC-MS/MS.
The method of claim 1, wherein the pre-treatment in 2) is performed by bringing the biological sample into contact with a mixed solution of methanol and strong acid or mixing the biological sample with a mixed solution of methanol and strong acid.
The method of claim 1, wherein the biological sample in 1) is blood.
The method of claim 1, wherein the strong acid in 2) is sulfuric acid, hydrochloric acid, or nitric acid.
The method of claim 1, wherein the mixed solution of methanol and strong acid in 2) comprises methanol and a strong acid mixed at a volume ratio of 9:1 ~ 8:2.
The method of claim 1, wherein 1) comprises:

a) obtaining 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 of claim 6, wherein eluting the biological sample in c) is performed by bringing formic acid into contact with the blood filter paper on which the biological sample was dropped.
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.