JP2023118717A - Analysis method and analyzer - Google Patents

Abstract

To quickly detect many more enzymes corresponding to each symptom of lysosomal diseases.SOLUTION: An analysis method includes: causing the ground substance of four or more enzymes having decreased or being lacking or being not normally functioning in the body of a patient of a lysosomal disease to react with one of the four or more enzymes that is included in a sample acquired from a living matter, thereby generating a reaction product; separating the reaction product by liquid chromatography; and detecting the separated reaction product by mass spectroscopy. The four or more enzymes include iduronic acid-2-sulfatase.SELECTED DRAWING: Figure 1

Description

The present invention relates to an analysis method and an analysis device.

In lysosomal storage diseases, enzymes that decompose proteins, fats, sugars, etc. are genetically reduced, defective, or not working properly. As a result, substances that should be degraded by lysosomes accumulate, resulting in cell death and organ damage. It is a disease that causes tissue dysfunction. For example, in mucopolysaccharidosis, glycosaminoglycan, which is a mucopolysaccharide, accumulates in lysosomes due to deficiency of hydrolase that works in lysosomes. Patients with mucopolysaccharidosis often die before reaching adulthood due to various symptoms such as bone and joint abnormalities and intellectual disability.

Lysosomal storage diseases are treated by enzyme replacement therapy, hematopoietic stem cell transplantation, and the like, but early detection is important to improve prognosis. Therefore, it has been proposed to conduct a screening test to examine whether the enzyme corresponding to the causative gene of lysosomal storage disease is sufficiently produced in newborns (see Non-Patent Document 1).

In such screening tests, substrates for enzymes associated with each lysosomal storage disease are added to a blood sample, and reaction products from the enzymatic reactions are analyzed using a tandem mass spectrometer or liquid chromatograph.
Detecting using a mass spectrometer and measuring the activity of the enzyme in vivo is performed (see Patent Document 1).

Japanese special table 2017-526923

Michael H. Gelb, C. Ronald Scott, and Frantisek Turecek. "New born screening for lysosomal storage diseases," Clinical chemistry, (USA), American Association for Clinical Chemistry, January 2015, Volume 61, Issue 2, pp. 335-346

However, in mass spectrometry, the substrate of the enzyme associated with lysosomal storage disease contained in the sample for mass spectrometry undergoes dissociation after being ionized, making it difficult to distinguish the dissociated ions from the reaction products. , there was a problem that the reaction products could not be sufficiently separated and detected.

An analysis method according to a preferred embodiment of the present invention comprises substrates for four or more enzymes that are reduced, defective, or not functioning normally in a patient's body in lysosomal storage diseases, and reacting an enzyme contained in a sample to produce a reaction product; separating the reaction product by liquid chromatography; and detecting the separated reaction product by mass spectrometry. The four or more enzymes comprise iduronate-2-sulfatase.
In a further preferred embodiment, said enzymes are 5 or more enzymes that are reduced, deficient or malfunctioning in said patient in a lysosomal storage disease.
In a further preferred embodiment, said enzymes comprise 4 or more enzymes that are reduced, deficient or malfunctioning in said patient in each of the 4 or more mucopolysaccharidosis subtypes.
In a further preferred embodiment, the enzyme comprises three or more enzymes selected from the group consisting of α-L-iduronidase, α-N-acetylglucosaminidase, N-acetylgalactosamine-6-sulfatase and N-acetylgalactosamine-4-sulfatase. Contains enzymes.
In a further preferred embodiment, said enzymes comprise 5 or more enzymes that are reduced, deficient or malfunctioning in the patient in each of the 5 or more mucopolysaccharidosis subtypes.
In a further preferred embodiment, said enzymes comprise α-L-iduronidase, α-N-acetylglucosaminidase, N-acetylgalactosamine-6-sulfatase and N-acetylgalactosamine-4-sulfatase.
In a further preferred embodiment, the solution containing the produced reaction product and the substrate is
It is introduced into a liquid chromatograph and the reaction product and the substrate are separated by the liquid chromatography.
In a further preferred embodiment, the retention time of at least one of said reaction products is shorter than the retention time of the corresponding substrate in said liquid chromatography.
In a further preferred embodiment, said at least one reaction product is iduronic acid-2-
It contains at least one enzymatic reaction product selected from the group consisting of sulfatase, N-acetylgalactosamine-6-sulfatase and N-acetylgalactosamine-4-sulfatase.
In a further preferred embodiment, in the liquid chromatography, the length of the column separating the reaction products is less than 50 mm.
In a further preferred embodiment the retention time is less than 3 minutes when said liquid chromatography is terminated.
A further preferred embodiment further comprises outputting information about an enzyme whose activity in the sample was lower than a reference value or the lysosomal storage disease corresponding to the enzyme among the enzymes.
An analyzer according to a preferred embodiment of the present invention comprises substrates for four or more enzymes that are reduced, defective, or not functioning normally in a patient's body, and one of the four or more enzymes obtained from the body in lysosomal storage disease. a sample introduction unit for introducing an analysis sample containing a reaction product obtained by reacting with an enzyme contained in the sample into a liquid chromatograph; and the reaction product contained in the introduced analysis sample. and a mass spectrometer that detects the reaction product separated by the liquid chromatograph by mass spectrometry, wherein the four or more enzymes include iduronate-2-sulfatase.
In a further preferred embodiment, said analytical sample comprises said reaction product and said substrate, and said liquid chromatograph separates said reaction product and said substrate contained in said introduced analytical sample.
In a further preferred embodiment, the output unit further comprises an output unit that outputs information about an enzyme whose activity in the sample is lower than a reference value, or information about the lysosomal storage disease corresponding to the enzyme.

According to the present invention, the activity of four or more enzymes associated with lysosomal storage diseases such as mucopolysaccharidosis can be rapidly measured.

FIG. 1 is a conceptual diagram for explaining the analysis method of one embodiment. FIG. 2 is a conceptual diagram showing a schematic configuration of an analyzer according to one embodiment. FIG. 3(A) is a graph schematically showing the detection intensity when the reaction product and the substrate are eluted from the liquid chromatograph at approximately the same time, and FIG. 3(B) shows the reaction product before the substrate. 4 is a graph schematically showing detection intensity when eluting from a liquid chromatograph. FIG. 4 is a flow chart showing the flow of the analysis method of one embodiment. FIG. 5 is a chromatogram when a sample containing no lysosomal storage disease-related enzymes was analyzed. FIG. 6 is a chromatogram when a sample containing lysosomal storage disease-related enzymes was analyzed. FIG. 7 is a chromatogram when a sample containing lysosomal storage disease-related enzymes was analyzed. FIG. 8 is a chromatogram when a sample containing lysosomal storage disease-related enzymes was analyzed.

Embodiments for carrying out the present invention will be described below with reference to the drawings. In the following embodiments, in each disease of lysosomal storage diseases, enzymes that are decreased or deficient in the patient's body due to genetic causes, or that do not work normally are referred to as lysosomal storage disease-associated enzymes. here,
"Disease" is defined correspondingly to each lysosomal storage disease-related enzyme, and shall refer to each disease type in mucopolysaccharidoses, for example.

FIG. 1 is a conceptual diagram for explaining the analysis method of this embodiment. In the analysis method of the present embodiment, substrates of lysosomal storage disease-related enzymes corresponding to a predetermined number of lysosomal storage diseases, particularly mucopolysaccharidoses, are reacted with the enzymes contained in the sample to obtain The reaction products are separated by liquid chromatography.

An operation is performed in which the lysosomal storage disease-associated enzymes contained in the sample Sp such as blood collected from the living body are reacted with the substrates of these lysosomal storage-associated enzymes. Here, the sample Sp is not particularly limited to blood, as long as the activity of the lysosomal storage disease-associated enzyme is statistically different between healthy subjects and lysosomal storage disease patients. In the following, the sample Sp is assumed to be collected from a human subject, but is not particularly limited. It is preferable that the subject be a child or a newborn under the age of 5 or under 3, in order to improve the prognosis of lysosomal storage disease by early detection.

In the following, the sample Sp is blood, and the dried blood spot (Dried Blood Spo
An example of reacting a sample Sp with a substrate using t; DBS) will be described. Since the whole blood can be used as it is, the dried blood spot has advantages such as the need for centrifugation is unnecessary, the operation is small, and the amount of blood to be collected is small. The sample Sp is dripped onto the filter paper C using a pipetting instrument or pipetting device such as a pipette P1 so that the entire detachable portion D of the filter paper C is soaked with the sample Sp. The sample Sp dropped onto the filter paper C is dried for several hours (arrow A11).

The dried sample Sp becomes a dried blood spot (DBS) on the filter paper C. The detachable portion D of the filter paper C is formed so that it can be easily removed from the body of the filter paper C by punching or the like. The detachable portion D is, for example, a disc-shaped portion with a diameter of 3 mm to 6 mm. When the removable portion D containing the sample Sp is removed, it is placed in a dispensing container W such as a dispensing tube or well plate (arrow A12).

A plurality of lysosome-associated enzyme substrates Sb and a predetermined amount (not shown) corresponding to the reaction product obtained from the substrate Sb by the enzymatic reaction are placed in the removable portion D containing the sample Sp and placed in the dispensing container W. of the internal standard is added. The internal standard is a substance for quantifying the ions corresponding to the reaction product by the detection intensity corresponding to the internal standard corresponding to the predetermined amount, and the reaction product obtained from the substrate Sb by the enzyme reaction. A substituted compound obtained by substituting a part of atoms with a stable isotope such as deuterium is appropriately used.
Instead of using the dried blood spot, the enzymatic reaction may be performed by bringing a solution obtained by dispensing a predetermined amount of blood into contact with the substrate solution.

After the substrate solution is added, the dispensing container W is incubated for an enzymatic reaction, such as several hours to several days. In the following embodiments, "enzymatic reaction" means lysosome-associated enzyme and substrate Sb
and "reaction solution" refers to the solution obtained by the enzymatic reaction. Thereafter, a component containing the reaction product P of the enzymatic reaction is extracted from the reaction solution to prepare an analysis sample Sa (
Arrow A13). The method for extracting the reaction product P is not particularly limited, but liquid-liquid extraction is preferable for protecting the analysis column of liquid chromatography, the ion source, etc., and the liquid-liquid extraction using ethyl acetate is preferred for extracting the substrate Sb in the analytical sample Sa. It is more preferable because the amount can be reduced. Substrate Sb of each lysosome-associated enzyme may remain in analysis sample Sa, and substrate S
The following explanation is given assuming that b is included.

The prepared analytical sample Sa is introduced into a liquid chromatograph, and the reaction product P and the corresponding substrate Sb are separated by liquid chromatography. An eluted sample eluted from the liquid chromatograph is introduced into the tandem mass spectrometer. Multiple reaction products P corresponding to different lysosomal storage disease-associated enzymes are separated by liquid chromatography and/or mass separation. The separated reaction product P is detected by the detector of the tandem mass spectrometer (arrow A14). Each reaction product P was analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/
MS) simultaneously. Here, "concurrently measured" means that a plurality of substances contained in the analysis sample Sa introduced into the liquid chromatograph at one time are separated and detected.

Lysosomal storage disease-related enzymes whose activities are measured by the analysis method of the present embodiment are lysosomal storage disease-related enzymes related to each disease type of mucopolysaccharidoses listed in Table 1 below (hereinafter referred to as mucopolysaccharidosis-related enzymes). It is preferred to include those selected from This determines whether the living body from which the sample Sp was collected has, or is suspected to have, a lysosomal storage disease corresponding to each lysosome-related enzyme, including any disease type of mucopolysaccharidosis. It can provide information for diagnosing whether or not Substrate Sb for lysosomal storage disease-associated enzymes to measure activity
is included in the substrate solution described above and added to the sample Sp.

The number of types of lysosomal storage disease-related enzymes whose activities are measured is preferably 4 or more, more preferably 5 or more. The more types of lysosomal storage disease-associated enzymes whose activities are measured, the more information for the diagnosis of lysosomal storage diseases can be rapidly provided. If there are too many types of lysosomal storage disease-related enzymes whose activities are measured, separation by liquid chromatography and mass spectrometry becomes difficult.
It is preferably set to 0 or less.

The lysosomal storage disease-related enzyme whose activity is measured by the analysis method of this embodiment is preferably selected from mucopolysaccharidosis-related enzymes. This can provide information for diagnosing whether the living body from which the sample Sp was collected is affected by each mucopolysaccharidosis, or whether it is suspected to be affected. In this case, the substrate Sb of the mucopolysaccharidosis-associated enzyme whose activity is to be measured
is included in the substrate solution described above and added to the sample Sp.

The mucopolysaccharidosis-related enzymes whose activities are measured by the analysis method of this embodiment are α-L-iduronidase, iduronate-2-sulfatase, α-N-acetylglucosaminidase, and N-acetylgalactosamine-6 listed in Table 1. - sulfatase and N-acetylgalactosamine-4-sulfatase. Thereby, the subject is a mucopolysaccharide selected from each mucopolysaccharidosis type I, type II, IIIB type, IV-A type and VI type, which has a higher incidence rate than other mucopolysaccharidosis disease types Information can be provided to diagnose whether one has, or is suspected of having, a subtype of the disease.

More preferably, the mucopolysaccharidosis-related enzyme whose activity is measured by the analysis method of this embodiment includes α-L-iduronidase and/or iduronate-2-sulfatase listed in Table 1. This determines whether or not the subject has, or is suspected to have, mucopolysaccharidosis type I and/or type II, which has a particularly high incidence rate compared to other disease types of mucopolysaccharidosis. It can provide information for diagnosing whether there is.

The mucopolysaccharidosis-associated enzyme whose activity is measured by the analysis method of this embodiment more preferably includes iduronate-2-sulfatase listed in Table 1. As a result, it is possible to determine whether or not the subject has mucopolysaccharidosis type II, which has a particularly high incidence in specific regions and genders compared to other mucopolysaccharidosis disease types. Information can be provided to diagnose whether there is suspicion. Mucopolysaccharidosis type II develops in humans due to X-chromosome recessive inheritance, so it often develops in males. Screening for male newborns preferably includes testing for mucopolysaccharidosis type II.

The mucopolysaccharidosis-related enzymes whose activities are measured by the analysis method of this embodiment are α-L-iduronidase, iduronate-2-sulfatase, α-N-acetylglucosaminidase, and N-acetylgalactosamine-6 listed in Table 1. -sulfatase and N-acetylgalactosamine-4-sulfatase, or most preferably including these five enzymes. As a result, the subject has each mucopolysaccharidosis I, which has a high incidence rate compared to other mucopolysaccharidosis disease types.
It can rapidly provide information to diagnose whether one has or is suspected of having Types II, IIIB, IV-A and VI.

…（１） The substrate Sb of the mucopolysaccharidosis-related enzyme used in the analysis method of the present embodiment is not particularly limited as long as the reaction product P can be separated by liquid chromatography. For example, one represented by the following chemical formula (1) is used. be able to.

…(1)

Examples of functional groups corresponding to X, R ₁ , R ₂ and n in the above chemical formula (1) are shown in Table 2 below.

FIG. 2 is a conceptual diagram showing the configuration of an analysis apparatus according to the analysis method of this embodiment. Analyzer 1
comprises a measurement unit 100 and a control unit 40 . The measurement unit 100 includes a liquid chromatograph 10
and a mass spectrometer 20 .

The liquid chromatograph 10 includes mobile phase containers 11a and 11b and liquid feed pumps 12a and 12b.
, a sample introduction part 13 , and an analysis column 14 . The mass spectrometer 20 has an ionization section 21
1, a first vacuum chamber 22a with an ion lens 221, a tube 212 for introducing ions from the ionization chamber 21 to the first vacuum chamber 22a, a second vacuum chamber 22b with an ion guide 222, and a third vacuum chamber 22c. The third vacuum chamber 22 c includes a first mass separator 23 , a collision cell 24 , a second mass separator 25 and a detector 30 .
The collision cell 24 has an ion guide 240 and a CID gas inlet 241 .

The information processing section 40 includes an input section 41 , a communication section 42 , a storage section 43 , an output section 44 and a control section 50 . The control unit 50 includes a device control unit 51 , an analysis unit 52 and an output control unit 53 .

A liquid chromatograph (LC) 10 utilizes the difference in affinity of each reaction product P and substrate Sb for the mobile phase and the stationary phase of the analytical column 14 to separate and differentiate each reaction product P and substrate Sb. Elute at the retention time. The type of the liquid chromatograph 10 is not particularly limited as long as the reaction product P and the substrate Sb can be separated with the desired accuracy such that the reaction product P can be detected by the mass spectrometer 20 . As the liquid chromatograph 10, nano LC, micro LC, high performance liquid chromatograph (HPLC), ultra high performance liquid chromatograph (UHPLC), etc. can be used.

The mobile phase containers 11a and 11b are provided with containers capable of storing liquids such as vials, and store mobile phases with different compositions. The mobile phases stored in mobile phase containers 11a and 11b are called mobile phase A and mobile phase B, respectively. The composition of the mobile phase A and mobile phase B is not particularly limited as long as the reaction product P and the substrate Sb can be separated with the desired accuracy. can.

Liquid-sending pumps 12a and 12b respectively send mobile phase A and mobile phase B at predetermined flow rates. Mobile phase A and mobile phase B respectively output from liquid-sending pumps 12 a and 12 b are mixed in the flow path and introduced into sample introduction section 13 . Liquid sending pump 12
A and 12b change the composition of the mobile phase introduced into the analysis column 14 over time by changing the flow rates of mobile phase A and mobile phase B, respectively.

Data indicating the composition of the mobile phase at each time from the time point corresponding to the start of analysis such as the introduction of the analysis sample Sa is called gradient data. The liquid feed pumps 12a and 12b are controlled based on the gradient data, and the mobile phase having the set composition is introduced into the analysis column 14. The time change of the composition of the mobile phase is not particularly limited as long as the reaction product P and the substrate Sb can be separated with the desired accuracy, but all of the reaction product P is eluted from the analysis column 14 with a retention time of less than 10 minutes. preferably less than 7 minutes, more preferably less than 3 minutes, even more preferably less than 2 minutes, even more preferably less than 1.5 minutes. The shorter the retention time of the reaction product P, the more quickly a large number of subjects, especially human neonates, can be screened for lysosomal storage diseases. The retention time when liquid chromatography is terminated by stopping is called analysis time. The analysis time can be set to 3 by adjusting the conditions of the analysis column 14, etc.
It has been found by the inventors that it can be less than a minute, more preferably less than 2.5 minutes. This allows efficient analysis. If the analysis time is too short, it becomes difficult to separate the reaction product P and the substrate Sb in liquid chromatography.

The sample introduction unit 13 includes a sample introduction device such as an autosampler, and introduces the analysis sample Sa into the mobile phase (arrow A1). The analysis sample Sa introduced by the sample introduction section 13 passes through a guard column (not shown) as appropriate and is introduced into the analysis column 14 .

The analysis column 14 has a stationary phase, and each reaction product P and substrate Sb contained in the introduced analysis sample Sa is detected by measuring the difference in affinity of each reaction product P and substrate Sb with respect to the mobile phase and the stationary phase. are used to elute at different times. The type of the analytical column 14 is not particularly limited as long as it can separate each reaction product P and the substrate Sb with the desired accuracy, but a reversed-phase column is preferable from the viewpoint of ease of handling and ease of ionization in mass spectrometry. . The stationary phase of the analytical column 14 is preferably silane with linear hydrocarbons such as C18 bound on a carrier such as silica gel.

The length of the analytical column 14 is preferably less than 50 mm, more preferably less than 40 mm, 3
0 mm or less is more preferable. The shorter the analysis column 14, the shorter the time required for analysis and the more efficient the analysis, which is preferable. If the analytical column 14 is too short, separation of the reaction product P and the substrate Sb in liquid chromatography becomes difficult, so the analytical column 14 is preferably longer than 3 mm, more preferably longer than 10 mm.
More preferably longer than 0 mm, even more preferably longer than 25 mm.

The analysis column 14 can elute the reaction product P obtained by cleaving the substrate Sb for each lysosomal storage disease-related enzyme before the elution of the substrate Sb is started, that is, with a short retention time. preferable.

FIG. 3A is a chromatogram schematically showing detection intensity in mass spectrometry when the reaction product P and the substrate Sb are eluted from the liquid chromatograph 10 substantially simultaneously, as a conventional example. In the following embodiments, as an example, ions separated as precursor ions are dissociated to generate product ions, and the product ions are separated and detected by multiple reaction monitoring (MRM) to detect the reaction product P Here is an example of detecting

After being ionized in the ionization section 21 of the mass spectrometer 20, the substrate Sb may become ions having m/z substantially equal to the m/z of the reaction product P due to in-source decomposition. In this case, ions corresponding to m/z of the reaction product P are mass-separated as precursor ions, and ions having m/z corresponding to fragment ions of the reaction product P are mass-separated and detected. FIG. 3A is the upper graph. In this chromatogram, the substrate Sb
is the peak Ps of the fragment ions of the in-source decomposed ions (hereinafter referred to as the peak Ps corresponding to the in-source decomposed substrate sb) and the peak Pp of the fragment ions of the reaction product P (hereinafter referred to as the reaction product P (called peak Pp corresponding to ) overlapped with peak P
p+Ps is observed.

Ions corresponding to the m/z of the substrate Sb are mass-separated as precursor ions, and the substrate Sb
The lower part of FIG. 3A is a chromatogram obtained by mass-separating and detecting ions having m/z corresponding to fragment ions of . As shown in this chromatogram, the fragment ion peak Psb of the substrate Sb is larger than the peak Pp corresponding to the reaction product P,
It can also be detected over a range of long retention times. Therefore, when the reaction product P elutes after the substrate Sb starts eluting from the liquid chromatograph 10, the reaction product P overlaps with the peak Ps corresponding to the in-source decomposed substrate Sb, and the reaction product P Detection cannot be performed accurately.

FIG. 3B is a chromatogram schematically showing the detection intensity in mass spectrometry when the reaction product P elutes from the liquid chromatograph 10 before the substrate Sb in the analysis method of the present embodiment. . Ions having m/z corresponding to the reaction product P and the substrate Sb are mass-separated as precursor ions to obtain fragment ions of the reaction product P and the substrate Sb.
Chromatograms obtained by mass-separating and detecting ions having m/z corresponding to fragment ions of are shown in the upper and lower parts of FIG. 3(B), respectively. Detection of the peak Psb of the fragment ions of the substrate Sb (lower part of FIG. 3(B)) and the detection of the peak Ps corresponding to the in-source decomposed substrate Sb substantially at the same time. gram (upper part of FIG. 3(B)). However, the peak Pp corresponding to the reaction product P
, does not overlap with the peak Ps corresponding to the in-source decomposed substrate Sb, and the reaction product P can be accurately detected and quantified.

Analytical column 14 is preferably iduronate-2-sulfatase, N-acetylgalactosamine-6-
A reaction product P of an enzymatic reaction of an enzyme containing at least one, more preferably at least two, and still more preferably three selected from the group consisting of sulfatase and N-acetylgalactosamine-4-sulfatase as a substrate of the enzymatic reaction It is eluted before the elution of Sb is started. Since the substrate Sb of these enzymes is particularly prone to in-source decomposition, the reaction product P can be detected more accurately by eluting a larger amount of the reaction product P of these enzymes prior to the corresponding substrate Sb. It can be carried out.
From the viewpoint of detecting the reaction product P more accurately according to the analysis conditions, the reaction product P of any lysosomal storage disease-associated enzyme can be eluted prior to the substrate Sb.

Returning to FIG. 2 , the eluted sample containing the reaction product P eluted from the analysis column 14 is introduced into the ionization section 21 of the mass spectrometer 20 . The eluate of the analysis column 14 is preferably input to the mass spectrometer 20 by online control without requiring operations such as dispensing by the user of the analysis device 1 (hereinafter simply referred to as "user").

The mass spectrometer 20 performs tandem mass spectrometry on the eluted sample introduced from the analytical column 14 to detect the reaction product P and the internal standard of the reaction product P. The path of the ionized elution sample Se is schematically indicated by a dashed-dotted arrow A2.
The type of mass spectrometer 20 and the method of mass spectrometry are not particularly limited as long as ions derived from each reaction product P can be detected with desired accuracy.

The ionization section 21 of the mass spectrometer 20 ionizes the elution sample Se containing the introduced reaction product P. The ionization method is not particularly limited as long as the reaction product P is ionized to the extent that the reaction product P can be detected with desired accuracy. A spray method (ESI) is preferred, and the following embodiments will also be described assuming that ESI is performed. The eluted sample Se, which is emitted from the ion source 211 and ionized, moves by a voltage applied to electrodes (not shown), passes through the tube 212, and enters the first vacuum chamber 22a.

The first vacuum chamber 22a, the second vacuum chamber 22b, and the third vacuum chamber 22c have a higher degree of vacuum in this order, and the third vacuum chamber 22c is evacuated to a high vacuum of, for example, 10 ⁻² Pa or less. . Ions incident on the first vacuum chamber 22a pass through the ion lens 221 and are introduced into the second vacuum chamber 22b. Ions entering the second vacuum chamber 22b pass through the ion guide 222 and are introduced into the third vacuum chamber 22c. The ions introduced into the third vacuum chamber 22c are
It is emitted to the mass separation section 23 . The ion lens 221 , the ion guide 222 and the like converge passing ions by electromagnetic action until they enter the first mass separation section 23 .

The first mass separation unit 23 selectively passes ions having m/z set by the electromagnetic action based on the voltage applied to the quadrupole as precursor ions and emits them toward the collision cell 24 . . The first mass separation unit 23 selectively allows the ionized reaction product P and the internal standard corresponding to the reaction product P to pass through as precursor ions.

The collision cell 24 performs collision induced dissociation (CID) while controlling the movement of ions by means of an ion guide 240.
dissociating the reaction product P ionized by and the internal standard corresponding to the reaction product P;
Generate fragment ions. A gas containing argon, nitrogen, or the like with which ions collide during CID (hereinafter referred to as CID gas) is introduced from the CID gas inlet 241 so as to have a predetermined pressure in the collision cell (arrow A3). . The generated fragment ions are emitted toward the second mass separation section 25 .

The second mass separation section 25 selectively passes fragment ions having m/z set by an electromagnetic action based on the voltage applied to the quadrupole and emits them toward the detection section 30 . The second mass separation unit 23 selectively allows the reaction product P and the internal standard fragment ions of the reaction product P to pass through.

In the mass spectrometer 20, when the substrate Sb shown in Table 2 is used, the reaction product P of the mucopolysaccharidosis-related enzyme corresponding to mucopolysaccharidosis type I falls within the range of 425.8 to 426.8. Separation by some m/z is preferred. More preferably, the reaction product P is separated by any m / z in the range of 425.8 to 426.8 as precursor ions, and the product ions are separated by any m in the range of 316.8 to 317.8 It is preferable to separate and detect by /z.

In the mass spectrometer 20, when the substrate Sb shown in Table 2 is used, the reaction product P of the mucopolysaccharidosis-related enzyme corresponding to mucopolysaccharidosis type II falls within the range of 643.9 to 644.9. Separation by some m/z is preferred. More preferably, the reaction product P is separated by any m / z in the range of 643.9 to 644.9 as precursor ions, and the product ions are separated by any m in the range of 358.9 to 359.9 It is preferable to separate and detect by /z.

When using the substrate Sb shown in Table 2 in the mass spectrometer 20, mucopolysaccharidosis IIIB
Reaction products P of mucopolysaccharidosis-related enzymes corresponding to the type preferably separate with m/z anywhere in the range 419.8-420.8. More preferably, the reaction product P is separated by any m / z in the range of 419.8 to 420.8 as a precursor ion,
It is preferable to separate and detect the product ions by any m/z in the range of 310.9 to 311.9.

In the mass spectrometer 20, when the substrate Sb shown in Table 2 was used, mucopolysaccharidosis IV-A
The mucopolysaccharidosis-related enzyme reaction product P corresponding to the type preferably separates with m/z anywhere in the range 684.9 to 685.9. More preferably, the reaction product P is separated by any m / z in the range of 684.9 to 685.9 as a precursor ion,
It is preferable to separate and detect the product ions by any m/z in the range of 372.7 to 373.7.

When the substrate Sb shown in Table 2 is used in the mass spectrometer 20, the reaction product P of the mucopolysaccharidosis-related enzyme corresponding to mucopolysaccharidosis type VI falls within the range of 656.9 to 657.9. Separation by some m/z is preferred. More preferably, the reaction product P is separated by any m / z in the range of 656.9 to 657.9 as precursor ions, and the product ions are separated by any m in the range of 344.9 to 345.9 It is preferable to separate and detect by /z.
The value of m/z selectively separated in the mass spectrometer 20 is appropriately set according to the ionized reaction product P and the fragment ions of the reaction product P to be detected, and is not limited to the above examples.

The detection unit 30 includes an ion detector such as a secondary electron multiplier or a photomultiplier, and detects fragment ions of the incident reaction product P and the internal standard of the reaction product P. The detection mode may be either a positive ion mode for detecting positive ions or a negative ion mode for detecting negative ions. A detection signal obtained by detecting fragment ions is converted to A/D by an A/D converter (not shown).
It is converted into a digital signal and input to the control section 50 of the information processing section 40 (arrow A4).
.

The information processing unit 40 includes an information processing device such as a computer, and serves as an interface with a user as appropriate, and performs processing such as communication, storage, and calculation of various data. Information processing unit 4
0 serves as a processing device for controlling the measurement unit 100 and performing analysis and display processing.
The information processing section 40 may be configured as one device integrated with the liquid chromatograph 10 and/or the mass spectrometer 20 . Also, part of the data used in the analysis method of this embodiment may be stored in a remote server or the like, and part of the arithmetic processing performed in the analysis method may be performed in a remote server or the like. The control of the operation of each unit of the measurement unit 100 may be performed by the information processing unit 40, or may be performed by each device constituting each unit.

The input unit 41 of the information processing unit 40 includes an input device such as a mouse, keyboard, various buttons and/or touch panel. The input unit 41 receives from the user information such as m/z values of ions to be detected, which are necessary for processing performed by the control unit 50 .

The communication unit 42 of the information processing unit 40 includes a communication device capable of communicating by wireless or wired connection via a network such as the Internet. The communication unit 42 receives data required for measurement by the measurement unit 100, transmits data processed by the control unit 50 such as analysis results of the analysis unit 52, and transmits and receives necessary data as appropriate.

The storage unit 43 of the information processing unit 40 includes a nonvolatile storage medium. The storage unit 43 stores measurement data output from the measurement unit 100, a program for the control unit 50 to execute processing, and the like.

The output unit 44 of the information processing unit 40 is controlled by the output control unit 53 and includes a display device such as a liquid crystal monitor and/or a printer, and outputs information related to measurement by the measurement unit 100,
The analysis results and the like of the analysis unit 52 are displayed on a display device or printed on a print medium and output.

The control unit 50 of the information processing unit 40 includes a processor such as a CPU. control unit 5
0 performs various processes by executing programs stored in the storage unit 43 or the like, such as controlling the measuring unit 100 and analyzing the measurement data output from the measuring unit 100 .

The device control unit 51 of the processing unit 50 controls the measurement operation of the measurement unit 100 based on the analysis conditions and the like set according to the input through the input unit 41 and the like. The device control unit 51 controls the liquid transfer pump 1
It controls the flow rates of 2a and 12b, controls the sample introduction by the sample introduction unit 13, and controls the m/z value of ions selectively passed through the first mass separation unit 23 and the second mass separation unit 25. equal.

The analysis unit 52 performs analysis such as quantification of the reaction product P based on the measurement data output from the measurement unit 100 . The analysis unit 52 acquires detection intensities corresponding to each reaction product P and its internal standard fragment ions from the measurement data output from the detection unit 30 and stores them in the storage unit 43 or the like.

The analysis unit 52 adds a known amount of the internal standard to the ratio obtained by dividing the detected intensity corresponding to the fragment ion of each reaction product P by the detected intensity corresponding to the fragment ion of the internal standard of the reaction product P. The multiplied value is calculated as the amount of each reaction product P. Based on the calculated amount of each reaction product P, the volume of the sample Sp such as the amount of blood, and the incubation time for the enzymatic reaction, the analysis unit 52 determines the amount of the sample Sp corresponding to each reaction product P. to calculate the activity of the enzyme. In addition, the analysis unit 52 creates data corresponding to a chromatogram in which retention times and detection intensities are associated with each other.

Based on the reference value stored in the storage unit 43, the analysis unit 52 determines whether the activity of each lysosomal storage disease-related enzyme is low to the extent that the subject has lysosomal storage disease or is suspected to have lysosomal storage disease. judge. The reference value is preferably set in advance based on the activity values of lysosome-associated enzymes in healthy subjects and lysosomal storage disease patients. The analysis unit 52 can make the above determination in consideration of variations in statistical values as appropriate.

Based on the measurement conditions of the measurement unit 100 and/or the analysis results of the analysis unit 52, the output control unit 53 determines the activity of each lysosome-related enzyme, the amount of each reaction product P, the chromatogram, whether the subject is afflicted, An output image including information about the suspected lysosomal storage disease is created and output to the output unit 44 . A doctor or the like can diagnose whether or not the subject is suffering from lysosomal storage disease based on the information included in the output image.

FIG. 4 is a flow chart showing the flow of the analysis method of this embodiment. step S100
In 1, a sample Sp such as blood is obtained from a subject such as a human newborn by a medical worker or the like. After step S1001 ends, step S1003 is started.

In step S1003, a substrate solution containing a predetermined number or more of substrates Sb for lysosomal storage disease-associated enzymes is prepared by a medical professional, a user, or the like. where the predetermined number is
For example, 4, 5, etc. After step S1003 ends, step S1005 is started. In step S1005, the lysosomal storage disease-associated enzyme and the substrate S contained in the sample Sp are brought into contact with the sample Sp and the substrate solution by a medical worker, user, or the like.
b is reacted to produce a reaction product P. After step S1005 ends, step S1007 is started.

In step S1007, an analytical sample Sa is prepared from a reaction solution containing a reaction product P and a substrate Sb by a medical professional, a user, or the like. After step S1007 ends, step S1009 starts. In step S1009, the sample introduction unit 13 introduces the analysis sample Sa into the liquid chromatograph 10, and the analysis column 14 separates the reaction product P and the substrate Sb. After step S1009 ends, step S1011 is started.

In step S1011, the mass spectrometer 20 separates and detects the separated reaction product P by mass spectrometry. After step S1011 ends, step S1013 starts. In step S1013, the analysis unit 52 analyzes whether the activity of the lysosome-associated enzyme in the sample Sp is reduced from the reference value based on the detected reaction product P data. After step S1013 ends, step S1015 is started.

In step S1015, the output unit 44 selects the sample S
enzymes with lower activity at p than the reference value or corresponding lysosomal storage diseases and/or
Alternatively, output information about the disease type of mucopolysaccharidosis. After step S1015 ends,
Processing is terminated.

According to the above-described embodiment, the following effects are obtained.
(1) The analysis device 1 according to the present embodiment comprises substrates Sb of four or more enzymes that are reduced, deficient, or not functioning normally in a patient's body in lysosomal storage diseases, and of these four or more enzymes, a sample introduction part into which the analysis sample Sa containing the reaction product P obtained by reacting with the enzyme contained in the sample Sp obtained from the Equipped with a liquid chromatograph 10 for separating the product P by liquid chromatography and a mass spectrometer 20 for detecting the reaction product P separated by the liquid chromatograph 10 by mass spectrometry, wherein the four or more enzymes are iduronic acid- Contains 2-sulfatase. As a result, the subject has each mucopolysaccharidosis type I, I
It can rapidly provide information for diagnosing whether or not the disease type selected from type IIB, type IV-A and type VI is afflicted or suspected of being afflicted. Furthermore, similar information can be quickly provided for mucopolysaccharidosis type II, which has a higher incidence in men and in specific regions.

(2) In the analysis apparatus according to the present embodiment, the analysis sample Sa includes the reaction product P and the substrate Sb, and the liquid chromatograph 10 includes the reaction product P and the substrate contained in the introduced analysis sample Sa. Sb is separated by liquid chromatography. This allows accurate detection of the substrate Sb even when in-source degradation occurs.

(3) The analysis device according to the present embodiment further includes an output unit 44 that outputs information about an enzyme whose activity in the sample Sp is lower than the reference value or lysosomal storage disease corresponding to the enzyme among the lysosome-associated enzymes. As a result, information for diagnosing lysosomal storage diseases can be quickly provided to doctors and the like.

The present invention is not limited to the contents of the above embodiments. Other aspects conceivable within the scope of the technical idea of the present invention are also included in the scope of the present invention.

(Example 1)
In the following examples, α-L-iduronidase, iduronate-2-sulfatase, α-N-
Experimental results are described in which the activities of five enzymes, acetylglucosaminidase, N-acetylgalactosamine-6-sulfatase and N-acetylgalactosamine-4-sulfatase, were assayed using the assay method described above.
In addition, the present invention is not limited to the numerical values and conditions shown in the following examples.

Reaction between sample and substrate A sample that does not contain the above five types of enzymes (control sample), a sample that contains the above five types of enzymes (specified concentration sample), and a sample obtained from healthy subjects ( sample from healthy subjects)
Each was dropped onto a filter paper and then dried to form a DBS-like spot. The spot was punched out into a disk shape with a diameter of 3 mm and placed in a 96-well plate. 30 μL of a substrate solution containing substrates for the above five types of enzymes and an internal standard of the reaction product P produced from the substrates by an enzymatic reaction is added to the wells in which the spots are arranged, and 37
°C for 16 hours.

Substrate and internal standard The substrates contained in the substrate solution were those shown in Table 2. The internal standard is the reaction product P
is shown in Table 2, and the following substitutes for the reaction product P shown in Table 2 were used.
Type I: A substitution of the mucopolysaccharidosis type I reaction product of Table 2 in which R ₂ is replaced with five deuterium atoms.
Type II: Substitution of the reaction product of mucopolysaccharidosis type II in Table 2 with five deuterium substitutions for _R2 .
Type IIIB: A substitution of the reaction product of mucopolysaccharidosis type IIIB of Table 2 with three deuterium substitutions for _R2 .
Type IV-A: A substitution product in which R ₂ of the reaction product of mucopolysaccharidosis type IV-A in Table 2 is replaced with five deuterium atoms.
Type VI: Substitution of the reaction product of mucopolysaccharidosis type VI of Table 2 with five deuterium substitutions for _R2 .

Preparation of Samples for Analysis After completion of the incubation, components containing reaction products were extracted from the resulting reaction solution using ethyl acetate. Ethyl acetate containing the extracted reaction product was placed in a separate container and then dried. then 55% water/45% acetonitrile to which 0.1% formic acid was added
The solution was added and used as a sample for analysis.

Liquid Chromatography Conditions An analytical sample was separated by liquid chromatography under the following conditions.
System: LCMS-8060 system (Shimadzu Corporation)
Analytical column: Kinetex XB-C18 (Phenomenex) (inner diameter 2.1 mm, length 150 mm, particle size 1.7 μm)
Injection volume: 1 μL
Column temperature: 40°C
Mobile phase:
(A) 0.1% formic acid (dissolved in water)
(B) 0.1% formic acid (dissolved in acetonitrile)
Flow rate: 0.4 mL/min
Gradient program:
Time (min) Concentration of mobile phase B (%)
0 30
0.5 30
3.5 100
5.0 100
5.01 30
6.0 Suspension

Conditions for Mass Spectrometry The eluted sample eluted in the above liquid chromatography was detected by tandem mass spectrometry directly connected to the elution port.
System: LCMS-8060 system (Shimadzu Corporation)
Ionization method: electrospray method, positive ion mode Measurement mode: multiple reaction monitoring (MRM)
temperature:
Desolvation line (DL) temperature: 250°C
Heat block temperature: 100°C
Interface temperature: -
Gas flow:
Nebulizer gas flow rate: 3 L/min
Drying gas flow rate: 5L/min
Heating gas flow rate: 15 L/min

The retention time of the eluted sample and the MRM measurement conditions (CE is collision energy voltage) are shown in Table 3 below.

A chromatogram of the control sample is shown in FIG. 5, a chromatogram of the sample at a predetermined concentration is shown in FIG. 6, and a chromatogram of the healthy subject sample is shown in FIG.
In the chromatograms corresponding to each mucopolysaccharidosis disease type, the upper row is the transitions corresponding to the reaction product, and the lower row is the chromatogram of the transitions (Table 3) corresponding to the internal standard. The peaks corresponding to insource-degraded substrates of types I, II, IIIB, IV-A and VI were designated P1s, P2s, P3s, P4s and P6s, respectively. The fragment ion peaks of the internal standards of type I, type II, type IIIB, type IV-A and type VI were designated as P1is, P2is, P3is, P4is and P6is, respectively. I
The peaks of the fragment ions of the type, type II, type IIIB, type IV-A and type VI reaction products were designated as P1p, P2p, P3p, P4p and P6p, respectively.

In the control sample, no reaction product fragment ion peaks were observed, but the peaks P2s, P3s, P4s and P4s corresponding to the in-source degraded substrates of types II, IIIB, IV-A and VI, respectively. P6s was observed (Fig. 5).

Type I, II, II
Fragment ion peaks P1p, P of reaction products of types IB, IV-A and VI
2p, P3p, P4p and P6p were observed. Of these, the fragment ion peaks P2p, P4p and P6p of the reaction products of types II, IV-A and VI have shorter retention times than the corresponding in-source degraded substrate peaks P2s, P4s and P6s. eluted. For five mucopolysaccharidosis-related enzymes, reaction products were separated and detected by liquid chromatography and mass spectrometry.

(Example 2)
The liquid chromatography conditions were changed as follows, and further, the mucopolysaccharidosis type I substrate was
A control sample (without enzyme) was subjected to LC/MS/MS under the same conditions as in Example 1, except that the reaction products were directly detected under mass separation conditions different from those used for mass separation.
Column: Kinetex XB-C18 (Phenomenex) (inner diameter 2.1 mm, length 30 mm, particle size 1.7 μm
m)
Gradient program:
Time (min) Concentration of mobile phase B (%)
0 30
0.01 30
1.30 100
1.50 100
1.51 30
2.50 Stop

8 is a diagram showing a mass chromatogram obtained in Example 2. FIG. Type I, Type II, II
peak P1s corresponding to insource-degraded substrates of types IB, IV-A and VI,
P2s, P3s, P4s and P6s were observed, respectively. Fragment ion peaks P1is, P2is of internal standards of types I, II, IIIB, IV-A and VI
, P3is, P4is and P6is were detected temporally separated from the substrate.
Therefore, it was shown that it is possible to separate reaction products and substrates by liquid chromatography even under the condition of an analysis time of 2.50 minutes.

DESCRIPTION OF SYMBOLS 1... Analyzer, 10... Liquid chromatograph, 14... Analysis column, 20... Mass spectrometer, 21
... ionization section, 23 ... first mass separation section, 24 ... collision cell, 25 ... second mass separation section,
30... Detecting unit 40... Information processing unit 50... Processing unit 51... Apparatus control unit 52... Analysis unit 5
3... Output control part, 100... Measurement part, C... Filter paper, D... Removable part, DBS... Dried blood spot, P... Reaction product, P1, P2... Pipette, P1is, P2is, P3is, P
4is, P6is: peaks of fragment ions of the internal standard of the reaction product, Pp, P1p
, P2p, P3p, P4p, P6p, peaks of fragment ions of reaction products, Ps,
P1s, P2s, P3s, P4s, P6s: Fragment ion peaks of ions resulting from in-source decomposition of substrate ions, Psb: Fragment ion peaks of substrate ions, Sa
... sample for analysis, Sb ... substrate, Sp ... sample.

An analysis method according to a preferred embodiment of the present invention comprises substrates for four or more enzymes that are reduced, defective, or not functioning normally in a patient's body in lysosomal storage diseases, and reacting an enzyme contained in a sample to produce a reaction product; separating the reaction product by liquid chromatography; and detecting the separated reaction product by mass spectrometry. In addition, separating by liquid chromatography includes separating by gradient elution using two types of mobile phases, and linear carbonization so that the retention time of the reaction product is shorter than the retention time of the corresponding substrate. and separating by a reverse-phase column packed with hydrogen-bonded silanes, wherein the four or more enzymes are α-L-iduronidase, iduronate-2-sulfatase, α-N-acetylglucosaminidase, N- 4 or more enzymes selected from the group consisting of acetylgalactosamine-6-sulfatase and N-acetylgalactosamine-4-sulfatase.
In a further preferred embodiment, said enzymes are 5 or more enzymes that are reduced, deficient or malfunctioning in said patient in a lysosomal storage disease.
In a further preferred embodiment, the enzyme comprises an enzyme that is reduced, deficient or malfunctioning in the patient for each of the 5 or more mucopolysaccharidosis subtypes.
In a further preferred embodiment, said enzymes comprise α-L-iduronidase, α-N-acetylglucosaminidase, N-acetylgalactosamine-6-sulfatase and N-acetylgalactosamine-4-sulfatase.
In a further preferred embodiment, the produced solution containing the reaction product and the substrate is introduced into a liquid chromatograph, and the reaction product and the substrate are separated by the liquid chromatography.
In a further preferred embodiment, isobutyl groups are introduced into the reversed-phase column.
In a further preferred embodiment, the reversed-phase column is packed with C18.
In a more preferred embodiment, one of the two mobile phases is 0.1% formic acid dissolved in water, and the other mobile phase is 0.1% formic acid dissolved in acetonitrile. It is characterized by being

Claims (15)

In lysosomal storage disease, substrates of 4 or more enzymes that are reduced, defective, or not functioning normally in the patient's body are reacted with an enzyme contained in a sample obtained from the living body among the 4 or more enzymes. , producing a reaction product;
separating the reaction products by liquid chromatography;
detecting the separated reaction products by mass spectrometry;
The analysis method wherein the four or more enzymes include iduronate-2-sulfatase. In the analysis method according to claim 1,
The assay method, wherein the enzymes are 5 or more enzymes that are reduced, deficient, or not functioning normally in the body of the patient in lysosomal storage disease. In the analysis method according to claim 1 or 2,
The assay method, wherein the enzymes are reduced, deficient, or not functioning normally in the body of the patient in each of the 4 or more disease types of mucopolysaccharidosis. In the analysis method according to claim 3,
The assay method wherein the enzyme comprises three or more enzymes selected from the group consisting of α-L-iduronidase, α-N-acetylglucosaminidase, N-acetylgalactosamine-6-sulfatase and N-acetylgalactosamine-4-sulfatase. In the analysis method according to claim 3 or 4,
The assay method, wherein the enzymes are reduced, deficient, or not functioning normally in the patient's body in each of the 5 or more disease types of mucopolysaccharidoses. In the analysis method according to claim 5,
The assay method wherein said enzymes include α-L-iduronidase, α-N-acetylglucosaminidase, N-acetylgalactosamine-6-sulfatase and N-acetylgalactosamine-4-sulfatase. In the analysis method according to any one of claims 1 to 6,
introducing a solution containing the produced reaction product and the substrate into a liquid chromatograph;
An analytical method wherein said reaction product and said substrate are separated by said liquid chromatography. In the analysis method according to claim 7,
In the liquid chromatography, the retention time of at least one of the reaction products is
Analytical methods shorter than the retention time of the corresponding substrate. In the analysis method according to claim 8,
The at least one reaction product includes a reaction product by at least one enzyme selected from the group consisting of iduronate-2-sulfatase, N-acetylgalactosamine-6-sulfatase and N-acetylgalactosamine-4-sulfatase. Analysis method. In the analysis method according to any one of claims 1 to 9,
In the liquid chromatography, the column for separating the reaction product has a length of 50 m
Analytical method that is less than m. In the analysis method according to any one of claims 1 to 10,
An analytical method in which the retention time at the end of said liquid chromatography is less than 3 minutes. In the analysis method according to any one of claims 1 to 11,
The analysis method further comprising outputting information about the enzyme whose activity in the sample is lower than the reference value or the lysosomal storage disease corresponding to the enzyme among the enzymes. In lysosomal storage disease, reaction between substrates of 4 or more enzymes that are reduced, defective, or not functioning normally in the patient's body and an enzyme contained in a sample obtained from the living body among the 4 or more enzymes a sample introduction unit for introducing an analysis sample containing the reaction product obtained by the reaction into the liquid chromatograph;
a liquid chromatograph that separates the reaction product contained in the introduced analytical sample;
A mass spectrometry unit that detects the reaction product separated by the liquid chromatograph by mass spectrometry,
The analytical device wherein said four or more enzymes comprise iduronate-2-sulfatase. The analysis device according to claim 13,
the analytical sample comprises the reaction product and the substrate;
The liquid chromatograph is an analysis device that separates the reaction product and the substrate contained in the introduced analysis sample. In the analysis device according to claim 13 or 14,
The analysis device further includes an output unit that outputs information about an enzyme whose activity in the sample is lower than a reference value, or information about the lysosomal storage disease corresponding to the enzyme.

Images