Liquid chromatography method and liquid chromatography system

Abstract

Provided are a method and a system for detecting hallucinogenic substances without using a mass spectrometer.

Description

Liquid chromatography method and liquid chromatography system

[0001] The present disclosure relates to liquid chromatography systems and methods, and in particular to systems and methods for detecting and / or quantifying active ingredients contained in psychedelic substances using liquid chromatography without the use of mass spectrometry.

[0002] Currently, there are no standard methods for analyzing and quantifying hallucinogenic compounds in food substances, such as hallucinogenic mushrooms. Gas chromatographs (GC), gas chromatograph mass spectrometers (GC-MS), liquid chromatographs (LC), liquid chromatograph mass spectrometers (LC-MS), and various other analytical devices are commonly used to test and analyze hallucinogenic compounds in samples. However, because some of the active compounds present in hallucinogenic mushrooms have similar structures and molecular weights, currently available analytical methods are often unreliable, especially when quantifying multiple different active compounds.

[0003] Previously, GC-MS and LC-MS have been used to identify such substances as illegal drugs and poisons (see, for example, Patent Document 1). In analyses using GC-MS or LC-MS, even if there are multiple compounds that cannot be completely separated by GC or LC, the mass spectrometer used as a detector can separate these components based on their mass-to-charge ratios and detect each component. The advantage of such analyses is that multiple active components can be reliably separated and each component can be accurately detected.

[0004] JP 2015-52570 A

[0005] However, compared to GC or LC, GC-MS and LC-MS are significantly more expensive and take significantly longer, resulting in lower throughput. Additionally, the additional training required to perform MS limits the number of people who can perform this analysis. Therefore, improved methods and systems for LC are needed to completely and reliably separate various hallucinogens from samples, thereby reducing analytical cost and complexity and achieving overall improvements.

[0006] In an effort to solve the problems discussed herein, the present disclosure describes methods and systems for detecting hallucinogens without the use of a mass spectrometer. Specifically, the methods and systems disclosed herein are useful for detecting and quantifying one or more hallucinogens, such as, for example, psilocybin, psilocin, norpsilocin, baeocystin, norbaeocystin, and aeruginascin, from a sample that may contain multiple of the hallucinogens.

[0007] In one aspect, the present disclosure describes a method for detecting psilocybin and / or psilocin in a sample without the use of a mass spectrometer. The method includes loading the sample onto a liquid chromatography column. Analytes in the sample are eluted, thereby isolating psilocybin and / or psilocin. The isolated psilocybin and / or psilocin are then detected.

[0008] In some embodiments, the detecting step includes measuring the retention time of a detection peak observed in the chromatogram. In some embodiments, the method may further include generating a detection peak based on the intensity and retention time measured by the detector for detecting the presence of psilocybin and / or psilocin. In this manner, the method may be used to detect psilocin and / or psilocybin, and may also be used to quantify the amount of psilocin and / or psilocybin in a given sample.

[0009] In some embodiments, in addition to detecting and / or quantifying the amount of psilocin and / or psilocybin in a given sample, the method may also be used to detect and / or quantify the amount of one or more of norpsirosin, baeocystin, norbaeocystin, and aeruginasin.

[0010] In some embodiments, during the elution, the rate of change in the mixing ratio of the solvents constituting the mobile phase of the sample during a first time period is greater than the rate of change in the mixing ratio of the solvents constituting the mobile phase during a second time period. During the second time period, the mixing ratio has a predetermined constant value. In such a mixing schedule, at least psilocybin is separated and sequentially eluted from the column, and at least psilocin is separated and sequentially eluted from the column during the second time period. In some embodiments, the mixing schedule can be appropriately modified so that norpsilocin and aeruginasin can be separated and sequentially eluted from the column during the first time period.

[0011] In some embodiments, during elution, the rate of change in the mixing ratio of the solvents constituting the mobile phase in a first period is less than the rate of change in the mixing ratio of the solvents constituting the mobile phase in a third period, and under such a mixing schedule, baeocystin and norbaeocystin are separated and sequentially eluted from the column.

[0012] The method may further include detecting the presence or absence of, and / or quantifying the amount of, each of psilocybin, psilocin, norpsilocin, baeocystin, norbaeocystin, and aeruginasin in the sample.

[0013] In another aspect, the present disclosure describes a detection system including a pump, a liquid chromatography column, and a processor. The processor is configured to control the pump to maintain a rate of change in a mixture ratio of solvents constituting a mobile phase of the sample during a first period after loading the sample containing one or more analytes at a rate greater than a rate of change in the mixture ratio of solvents constituting the mobile phase during a second period. During the second period, the mixture ratio is maintained at a predetermined constant value.

[0014] In another aspect, the present disclosure describes a non-transitory storage medium operatively coupled to a processor and having stored thereon instructions that, when executed by the processor, cause the processor to control a pump of a detection system coupled to a liquid chromatography column such that, after loading a sample containing one or more analytes, a rate of change in a mixture ratio of solvents constituting a mobile phase of the sample during a first time period is maintained greater than a rate of change in the mixture ratio of solvents constituting the mobile phase during a second time period, wherein the mixture ratio is maintained at a predetermined constant value during the second time period.

[0015] Additional features and advantages of the subject technology will be set forth in the description that follows, and in part will be obvious from the description, or may be learned by the practice of the subject technology. The advantages of the subject technology may be realized and attained by the structure particularly pointed out in the description, as well as the embodiments herein and the accompanying drawings.

[0016] It is to be understood that both the foregoing general description and the following detailed description are presented for purposes of illustration and description and are intended to provide further explanation of the subject technology.

[0017] Various features of illustrative embodiments of the invention are described below with reference to the drawings. The illustrated embodiments are intended to illustrate, but not to limit, the invention. The drawings include the following figures:

[0018]

[0023] Figure 1 is a schematic diagram of one embodiment of a liquid chromatography system for practicing a method according to the present disclosure.

[0024] Figure 2 shows a plot of mixing ratio values ​​over a period of time based on a mixing schedule according to one embodiment of the present disclosure.

[0025] Figure 3 shows a graph of an example chromatogram measurement obtained from a measurement performed on a sample using the mixing schedule described in the inset.

[0026] Figure 4 shows a graph of the quantification of various hallucinogenic components in a sample determined after implementation of the mixing schedule shown in Figure 3 and described herein.

[0027] Figure 5 shows a graph of the quantification of various hallucinogenic components in a sample determined after implementation of the mixing schedule shown in Figure 3 and described herein.

[0028] Figure 6 shows a graph of the quantification of various hallucinogenic components in a sample determined after implementation of the mixing schedule shown in Figure 3 and described herein.

[0029] Figure 7 shows a graph of the quantification of various hallucinogenic components in a sample determined after implementation of the mixing schedule shown in Figure 3 and described herein.

[0029] Figure 8 shows a graph of the quantification of various hallucinogenic components in a sample determined after implementation of the mixing schedule shown in Figure 3 and described herein. 1 shows a graph of an example chromatogram measurement obtained by measurements performed on a sample using the mixing schedule illustrated in the inset of the figure.

[0019] While various configurations of the subject technology have been shown and described in illustrative form in this disclosure, it is understood that these configurations will be readily apparent to those skilled in the art. As will be seen from the following description, other and different configurations of the subject technology are possible, and several details may be modified in various other respects, all without departing from the scope of the subject technology. Accordingly, the summary, drawings, and detailed description are to be regarded as illustrative in nature, and not as limiting.

[0020] The detailed description set forth below is intended to describe various configurations of the subject technology, and is not intended to describe the limited configurations in which the subject technology may be practiced. The accompanying drawings are incorporated herein by reference and constitute a part of the detailed description. The detailed description includes specific details to provide a thorough understanding of the subject technology. However, it will be apparent to those skilled in the art that the subject technology may be practiced without such specific details. In some instances, well-known structures and components are shown in block diagram form to avoid obscuring the concepts of the subject technology. For ease of understanding, similar components are designated with the same element numbers.

[0021] Although terms including ordinal numbers such as "first" and "second" may be used to describe various components, the components are not limited by these terms and are used solely to distinguish one component from another.

[0022] Throughout this specification, terms such as "comprises," "comprising," "include," "containing," "has," "having," and the like, or derivatives thereof, are understood to be inclusive and thus specify the presence of stated features, integers, steps, operations, elements, ingredients / components, or combinations thereof, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, ingredients / components, or combinations thereof. Thus, unless expressly stated otherwise, "comprises / include" and derivatives thereof, such as "comprises / comprising / include," are understood to imply the inclusion of stated elements, rather than the exclusion of any other elements.

[0023] In one aspect of the present disclosure, a system for detecting and / or quantifying hallucinogens without the use of a mass spectrometer is described. The system may include a pump, a liquid chromatography column, and a processor configured to control the pump. The system may generally be divided into various units, such as a measurement unit, a data processing unit, a control unit, and an operation unit.

[0024] In some embodiments, the measurement unit may include a high-performance liquid chromatography (HPLC) instrument. Typically, an HPLC includes one or more mobile phase containers containing one or more solvents that constitute the mobile phase to be applied to a liquid chromatography column. The measurement unit may also include a pump for drawing solvents from the one or more mobile phase containers and applying the drawn-up solvents to the column. In some embodiments, the measurement unit may further include a mixer coupled to the mobile phase containers for mixing the mobile phases at a predetermined mixing ratio. In some embodiments, the mixer may include, for example, a plurality of variable-opening solenoid valves that can be opened or closed to draw an appropriate amount of solvent from the mobile phase container.

[0025] In some embodiments, the measurement unit may also include an injection unit that can inject a liquid sample into the mobile phase drawn from the mobile phase container (optionally after appropriately mixing the solvents in a desired ratio) before injecting this (optionally mixed) mobile phase into the column. In some embodiments, the injection unit may be programmable so that the user can determine the amount of liquid sample to inject into the mobile phase.

[0026] A pump can then load the mobile phase and sample (optionally suitably mixed) onto the column, which is configured to temporally separate the various components (e.g., active ingredient, other compounds, and impurities) in the injected liquid sample. This process of temporally separating the components of a liquid sample is commonly referred to as elution, and the separated components are typically referred to as the eluate.

[0027] In some embodiments, the measurement unit may also include a column oven or thermostat designed and constructed to control the temperature of the column, thus maintaining the column at a particular temperature.

[0028] In some embodiments, the measurement unit further includes a detector for detecting one or more of the eluates eluting from the column. As used herein, the term "detecting" refers to determining whether a particular molecule or chemical is present. In some embodiments, the detector may be an analytical detector, such as an ultraviolet analytical detector (UV detector) or an ultraviolet-visible analytical detector (UV-Vis detector). In some embodiments, the detector may be a photodiode array detector. In some embodiments, the detector may be any suitable detector capable of detecting components in the solution phase, since the mobile phase includes a non-volatile solution containing phosphoric acid. On the other hand, detectors that require vaporization of the eluate are not suitable for the present methods and / or systems.

[0029] Typically, a detector may include a light source capable of generating light of an appropriate wavelength, a detection cell, and a light sensor. Light from the light source passes through a liquid in the detection cell, and the intensity of the light transmitted through the liquid is measured by the light sensor. The intensity of the transmitted light at a particular wavelength indicates the absorbance of the light at that wavelength.

[0030] In some embodiments, the measurement unit may also include an analog-to-digital converter for converting the detection signal into digital data, for example, by sampling the detection signal from the detector at predetermined sampling time intervals.

[0031] The data processing unit may include, as its functional blocks, a data collection section, an active ingredient identification processor, and an active ingredient amount determination processor. Without wishing to be bound by a particular theory, the amount of absorbance of light at a particular wavelength may indicate the presence of a particular molecule in a liquid. Therefore, by measuring the absorbance, it is possible to determine whether a molecule is present or not. The active ingredient identification processor may process the detection signal (in analog or digital form) to determine whether one, two, three, four, five, six, or more active ingredient molecules are present in the eluate.

[0032] In some embodiments, a calibration curve can be obtained by determining the area under the curve of absorbance versus time for various known concentrations of a specific molecule. A calibration curve allows for quantification of the specific molecule in a given sample whose concentration is unknown. Thus, the active ingredient amount determination processor can process the detection signal (in analog or digital format) to determine the amount of one, two, three, four, five, six, or more active ingredient molecules present in the eluate. For example, in embodiments using the system to detect and / or quantify specific hallucinogens, a calibration curve can be obtained by using a control solution as the sample. Thus, in some embodiments, the sample can be a control solution containing only a predetermined amount of the control analyte. In some embodiments, the control analyte can be psilocybin. In some embodiments, the control analyte can be psilocin. In some embodiments, the control analyte can be norpsirosin. In some embodiments, the control analyte can be baeocystin. In some embodiments, the control analyte can be norbaeocystin. In some embodiments, the control analyte may be aeruginasin. Thus, a calibration curve for the amount of the control analyte may be prepared by using a control solution and controlling the mixing schedule using a hallucinogenic assay control program to elute, detect, and quantify the corresponding control analyte in the control solution by liquid chromatography, and comparing the amount with the known amount in the control solution.

[0033] The control unit may include one or more controllers configured to control various components of the measurement unit, such as the mixer, pump, detector, and data processing unit, to initiate sample processing, initiate measurements, and / or perform other operations. In some embodiments, the control unit may control the pump and / or mixer to achieve a specific mixing ratio of solvents in the mobile phase from the mobile phase reservoir before adding the sample. Furthermore, in some embodiments, the control unit may change the mixing ratio at a predetermined rate. In some embodiments, the control unit may change the rate of change of the mixing ratio.

[0034] In some embodiments, the control unit can control the flow rate of the mobile phase (including the sample) through the column. In some embodiments, the flow rate can be any value within a range of about 100 μl / min to about 10 ml / min, e.g., about 100 μl / min to about 150 μl / min, about 100 μl / min to about 200 μl / min, about 100 μl / min to about 300 μl / min, about 100 μl / min to about 400 μl / min, about μl / min to about 500 μl / min, about 100 μl / min to about 600 μl / min, about 100 μl / min to about 700 μl / min, or about 100 μl / min to about 800 μl / min. l / min, about 100 μl / min to about 900 μl / min, about 100 μl / min to about 1000 μl / min, about 200 μl / min to about 300 μl / min, about 200 μl / min to about 400 μl / min, about 200 μl / min to about 500 μl / min , about 200 μl / min to about 600 μl / min, about 200 μl / min to about 700 μl / min, about 200 μl / min to about 800 μl / min, about 200 μl / min to about 900 μl / min, about 200 μl / min to about 1000 μl / min, about 30 0 μl / min to about 500 μl / min, about 300 μl / min to about 750 μl / min, about 300 μl / min to about 1000 μl / min, about 400 μl / min to about 800 μl / min, about 500 μl / min to about 1000 μl / min, about 500 μl / min l / min to about 1000 μl / min, about 500 μl / min to about 1.1 ml / min, about 500 μl / min to about 1.2 ml / min, about 500 μl / min to about 1.3 ml / min, about 500 μl / min to about 1.4 ml / min, about 500 μl / min The flow rate may be between about 1.5 ml / min, about 500 μl / min and about 2.0 ml / min, about 1 ml / min and about 2 ml / min, about 1 ml / min and about 3 ml / min, about 1 ml / min and about 4 ml / min, about 1 ml / min and about 5 ml / min, about 1 ml / min and about 6 ml / min, about 1 ml / min and about 7 ml / min, about 1 ml / min and about 8 ml / min, about 1 ml / min and about 9 ml / min, about 1 ml / min and about 10 ml / min, or any range overlapping any two of the above-mentioned ranges.

[0035] In some embodiments, the system may include two mobile phase containers containing two different solutions, which may also be referred to as mobile phase A and mobile phase B. The control unit may control the mixing ratio (volume ratio) of mobile phase B to mobile phase A. In some embodiments, the mixing ratio may be in the range of about 0.01% to about 99.99%. For example, the mixing ratio may be about 0.01% to about 0.05%, about 0.01% to about 1%, about 0.01% to about 5%, about 0.01% to about 10%, about 0.01% to about 15%, about 0.01% to about 15%, about 0.01% to about 20%, about 0.01% to about 25%, about 0.01% to about 30%, about 0.01% to about 35%, about 0.01% to about 40%, about 0.01% to about 55%, about 0.01% to about 60%, about 0.01% to about 65%, about 0.01% to about 70%, about 0.01% to about 75%, about 0.01% to about about 80%, about 0.01% to about 85%, about 0.01% to about 90%, about 0.01% to about 95%, about 0.01% to about 99.99%, about 1% to about 5%, about 1% to about 10%, about 1% to about 15%, about 1% to about 15%, about 1% to about 20%, about 1% to about 25%, about 1% to about 30%, about 1% to about 35%, about 1% to about 40%, about 1% to about 55%, about 1% to about 60%, about 1% to about 65%, about 1% to about 70%, about 1% to about 75%, about 1% to about 80%, about 1% to about 85%, about 1% to about 90%, about 1% up to about 95%, about 1% to about 99.99%, about 5% to about 10%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 60%, about 5% to about 70%, about 5% to about 80%, about 5% to about 90%, about 5% to about 99.99%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 99.99%, about 20% to about 3 0%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 99.99%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 99.99%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% to about 99.99%,It may be any value within the ranges of about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 99.99%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 99.99%, about 70% to about 80%, about 70% to about 90%, about 70% to about 99.99%, about 80% to about 90%, about 80% to about 99.99%, about 90% to about 99.99%, or any range overlapping any two of the above-mentioned ranges.

[0036] 10%, 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99.99%, or any value between any two of these values.

[0037] The mixing ratio may be varied, and the rate of change of the mixing ratio may be controlled. The rate of change of the mixing ratio of mobile phase B to mobile phase A may be in the range of about 1% / min to about 99% / min. Thus, for example, the rate of change of the mixing ratio may be about 1% / min, about 5% / min, about 10% / min, about 15% / min, about 20% / min, about 25% / min, about 30% / min, about 35% / min, about 40% / min, about 45% / min, about 50% / min, about 55% / min, about 60% / min, about 65% / min, about 70% / min, about 75% / min, about 80% / min, about 85% / min, about 90% / min, about 95% / min, about 99% / min, any value between any two of these values, or any value within any range between and including any two of these values.

[0038] Also, in some embodiments, the rate of change of the mixing ratio may or may not be constant over a given period of time. In other words, the rate of change of the mixing ratio may increase over a first period of time, remain constant over a second period of time, and decrease over a third period of time. Thus, for example, the rate of change of the mixing ratio may increase from about 5% / min to about 50% / min over a period of time, e.g., about 5 minutes, or decrease from about 95% / min to about 0% / min over a period of time (e.g., 1 minute) (i.e., the mixing ratio may not change). The increase and / or decrease may or may not be monotonic. For example, the rate of change of the rate of change of the mixing ratio may vary over a period of time.

[0039] In some embodiments, the control unit may implement a mixing schedule having several time periods with different rates of change of the mixing ratio. For example, the control unit may implement a mixing schedule in which the mixing ratio changes from a first mixing ratio to a second mixing ratio over a first period (e.g., 1 minute), remains constant at the second mixing ratio for a second period (e.g., 5 minutes), changes from the second mixing ratio to a third mixing ratio over a third period (e.g., 30 seconds), and changes from the third mixing ratio to a fourth mixing ratio over a fourth period (e.g., 1 minute). Thus, the control unit may implement any suitable schedule having any suitable number of periods during which the rate of change of the mixing ratio varies or remains constant.

[0040] For example, in one embodiment, the control unit may execute a schedule in which the mixing ratio during the second time period remains constant at a predetermined rate, and the rate of change of the mixing ratio during the first time period is greater than the rate of change of the mixing ratio during the second time period. In other words, the control unit may execute a schedule in which the mixing ratio changes during the first time period and remains constant during the second time period. It is contemplated that the rate of change of the mixing ratio during the first time period need not be constant, as long as it is non-zero. Furthermore, it is contemplated that the rate of change of the mixing ratio during the first time period may increase or decrease monotonically or at a varying rate.

[0041] In a further specific embodiment, the control unit may execute a schedule in which the mixing ratio remains constant at a predetermined rate during the second time period, the rate of change of the mixing ratio during the first time period is greater than the rate of change of the mixing ratio during the second time period, and the rate of change of the mixing ratio during the third time period is greater than the rate of change of the mixing ratio during the first time period. Thus, for example, the mixing ratio may change from 1 to a predetermined constant (e.g., 95%) during the first time period, remain constant at the predetermined constant value during the second time period, and then decrease to 1 during the third time period, where the first time period is longer than the third time period. In some embodiments, the mixing ratio may change to a first predetermined value at a first rate during the first time period, then change from the first predetermined value to a second predetermined value at a third rate faster than the first rate during the third time period, and then remain at the second predetermined value during the second time period. Furthermore, in the above-described mixing schedule, each period may be divided into multiple sub-periods, and the rate of change of the mixing ratio in each of these sub-periods may be constant, or the rate of change of the mixing ratio may differ from one sub-period to another.

[0042] Without being bound by theory, different components in a sample may elute during different periods in the mixing schedule for varying amounts of time depending on the concentration of that particular component in the sample.

[0043] The operational unit is configured to allow an operator (user) to instruct the control unit to initiate measurements or perform other operations. In some embodiments, the operational unit may include a user interface, such as one or more physical buttons, one or more software buttons, a display, and / or other means, by which a user can interact with the control unit to change one or more parameters related to the performance of the system for detecting and / or quantifying a sample. In some embodiments, the user interface may include a display capable of visually displaying the results of measurements and analyses and configured to display other information related to the operation of the system.

[0044] It is generally understood that a processor can include, for example, a computer, program logic, or other substrate configuration representing data and instructions that operate as described herein. In other embodiments, a processor can include controller circuitry, processor circuitry, processors, general-purpose single-chip or multi-chip microprocessors, digital signal processors, embedded microprocessors, microcontrollers, and the like.

[0045] It will further be appreciated that in one embodiment, program logic may advantageously be implemented as one or more components, which may advantageously be configured to execute on one or more processors, including, but not limited to, software or hardware components, modules such as software modules, object-oriented software components, class and task components, processes, methods, functions, attributes, procedures, subroutines, segments of program code, drivers, firmware, microcode, circuits, data, databases, data structures, tables, arrays, and variables.

[0046] It is contemplated that the various units disclosed herein (e.g., processing unit, control unit, operation unit, display unit, etc.) may be integrated into fewer units. Also, any one of these units may be divided into multiple units. The described units may be implemented as hardware, software, firmware, or any combination thereof. Furthermore, the described units may reside in separate locations connected by a wired or wireless network or the Internet.

[0047] The following description provides a specific example of one embodiment of the system described herein.

[0048] 1 is a schematic diagram of an exemplary LC system that can be used to implement the methods disclosed herein for mass spectrometer-free detection of hallucinogens in a sample, which may include one or more of psilocybin, psilocin, norpsilocin, baeocystin, norbaeocystin, and aeruginasin.

[0049] The molecular formula of the substance described above is shown here:

[0050]

[0051]

[0052] As shown in FIG. 1, the LC system includes a measurement unit 1 , a data processing unit 2 , a control unit 3 , an operation unit 4 , and a display unit 5 .

[0053] The measurement unit 1 is a high-performance liquid chromatograph (HPLC) and includes mobile phase containers 11A and 11B, a mixer 12, a liquid delivery pump 13, an injection unit 14, a column 15, a column oven 16, a UV detector 17, and, optionally, an ADC 18. The mobile phase containers 11A and 11B contain different mobile phases (hereinafter referred to as "mobile phases A and B"). The mixer 12 is connected to the mobile phase containers 11A and 11B and may include, for example, multiple variable-opening solenoid valves for mixing the two mobile phases A and B at a predetermined mixing ratio. The liquid delivery pump 13 draws the mobile phases A and B from the mobile phase containers 11A and 11B via the mixer 12 and supplies them to the column 15. Before injecting the mobile phase into the column 15, the injection unit 14 injects a certain amount of liquid sample into the mobile phase supplied by the liquid delivery pump 13. The mobile phase (comprising a mixture of mobile phases A and B) and sample are then loaded onto column 15. Column 15 temporally separates components (e.g., active ingredients, other compounds, and impurities) in the injected liquid sample. Column oven 16 controls the temperature of column 15. Ultraviolet analytical detector (UV detector) 17 detects one or more components in the eluate emerging from the outlet port of column 15. Analog-to-digital converter (ADC) 18 samples the detection signal from UV analytical detector 17 at predetermined sampling time intervals and converts the signal into digital data.

[0054] The data processing unit 2 includes, as its functional blocks, a data collection section 21, an active ingredient identification processor 22, and an active ingredient amount determination processor 23. This unit processes the detection data obtained by the ultraviolet analysis detector 17 (and digitized by the ADC 18) to identify active ingredients in the liquid sample and determine their amounts. The controller 3 may include a memory (e.g., one or more non-volatile storage devices) in which a hallucinogenic analysis control program 31 is stored. This program causes the CPU and other devices in the control unit 3 to appropriately control the relevant sections of the measurement unit 1 and the data processing unit 2 to perform the analysis operations described below. The operation unit 4 allows an operator (user) to command the control unit 3 to start measurements and perform other operations. The display unit 5 is used to display analysis results and other types of information.

[0055] In some embodiments, the hallucinogenic analysis control program 31 may control the mixer 12 and pump 13 to achieve a particular mixing ratio (volume to volume) of mobile phase B to mobile phase A, a particular rate of change of the mixing ratio, a particular rate of change of the rate of change of the mixing ratio, and a flow rate of the mobile phase as it is loaded onto the column 15. Thus, the hallucinogenic analysis control program 31 may control the mixer 12 and pump 13 to implement a particular mixing schedule in which the mixing ratio and / or rate of change of the mixing ratio may be varied according to a specified schedule.

[0056] In one embodiment, hallucination analysis control program 31 implements a schedule in which the mixture ratio increases from 1 to 95% over a first period of approximately 13 minutes, remains constant at 95% for a second period of approximately 40 seconds, and decreases to 1 over a third period of approximately 20 seconds. An example schedule is shown in Figure 2, which shows a plot of mixture ratio (Y-axis) over time (X-axis).

[0057] In some embodiments, mobile phase A contained in mobile phase container 11A may include an aqueous solution of phosphoric acid. Mobile phase A may further include a component such as sodium perchlorate monohydrate. In some embodiments, the concentration of phosphoric acid in mobile phase A may be in the range of 0.01% to 0.5%, for example, in the range of about 0.5% to about 0.1%. In some embodiments, mobile phase B contained in mobile phase container 11B may include phosphoric acid dissolved in acetonitrile. The concentration of phosphoric acid in mobile phase B may be in the range of 0.01% to 0.5%, for example, in the range of about 0.5% to about 0.1%. In one specific embodiment, the concentration of the aqueous phosphoric acid solution in mobile phase A is 0.085% (v / v). The concentration of the phosphoric acid-containing acetonitrile in mobile phase B is also 0.085% (v / v).

[0058] Column 15 is an OSD column for reversed-phase LC. In a specific embodiment, the column has a length (L) of 150 mm and an inner diameter (ID) of 4.6 mm. The particle size of the packing material is 3 μm. The particle pore size is 8 nm. As a specific example, a column manufactured by Shimadzu Corporation under the trade name "Shim-Pack Scepter C18-120" can be used as column 15.

[0059] In some embodiments, UV detector 17 may include a detection cell through which the eluate from column 15 is passed. A UV light source (not explicitly shown) is used to generate light of a predetermined wavelength, which is passed through the eluate in the detection cell. The intensity of the light passed through the eluate is detected to provide a detection signal indicative of the absorbance of light by the eluate. In some embodiments, UV detector 17 may be an ultraviolet-visible (UV-Vis) analytical detector.

[0060] In some embodiments, the detection wavelength is in the range of about 200 nm to about 280 nm. In other words, in some embodiments, the UV light source in UV detector 17 generates ultraviolet light having a wavelength in the range of about 200 nm to about 280 nm. In some embodiments, the detection signal is obtained at a wavelength of about 220 nm. In some embodiments, the detection signal is obtained at a wavelength of about 254 nm.

[0061] In some embodiments, the sample may comprise a fluid sample containing one, two, three, four, five, or all of psilocybin, psilocin, norpsilocin, baeocystin, norbaeocystin, and aeruginasin. In some embodiments, the sample may be a plasma sample. In some embodiments, the sample may be a human plasma sample.

[0062] In one aspect of the present disclosure, a non-transitory computer-readable medium is provided. The non-transitory computer-readable medium has instructions stored thereon and is coupled to a processor. When executed by the processor, the instructions cause a hallucinogenic analysis control program to execute an appropriate mixing schedule. Thus, in some embodiments, when executed by the processor, the instructions cause the processor to control a pump of a detection system (e.g., a detection system disclosed herein) coupled to a liquid chromatography column (e.g., column 15) so that a rate of change in the mixing ratio of solvents constituting a mobile phase of the sample during a first time period after loading the sample containing one or more analytes is maintained at a rate greater than a rate of change in the mixing ratio of solvents constituting the mobile phase during a second time period. During the second time period, the mixing ratio is maintained at a predetermined constant value.

[0063] In one aspect of the present disclosure, a method for detecting an active ingredient in a sample without using a mass spectrometer is described. The active ingredient may be, for example, a hallucinogen. In some embodiments, the hallucinogen may be one, two, three, four, five, or all of psilocybin, psilocin, norpsilocin, baeocystin, norbaeocystin, and aeruginasin.

[0064] The method includes loading a sample onto a liquid chromatography column, isolating an active component by eluting an analyte in the sample (e.g., by performing liquid chromatography), and then detecting the active component. Thus, in some embodiments, the method may include loading a sample onto a liquid chromatography column, isolating psilocybin and / or psilocin by eluting an analyte in the sample (e.g., by performing liquid chromatography), and then detecting psilocybin and / or psilocin. In some embodiments, the detecting step includes determining whether psilocybin and / or psilocin are present.

[0065] In some embodiments, the detecting step includes measuring the retention time of a detection peak observed in the chromatogram. In some embodiments, the method may further include generating a detection peak based on the intensity and retention time measured by the detector for detecting the presence of psilocybin and / or psilocin.

[0066] In some embodiments, the methods are capable of quantifying the amount of psilocybin and / or psilocin present in a sample.

[0067] In some embodiments, the analyte in the sample further comprises one, two, three, or four of norsyrosin, baeocystin, norbaeocystin, and aeruginasin. Thus, in some embodiments, the method further comprises detecting at least one of norsyrosin, baeocystin, norbaeocystin, and aeruginasin. For example, the method may further comprise detecting norsyrosin. In some embodiments, the method may further comprise detecting baeocystin. In some embodiments, the method may further comprise detecting norbaeocystin. In some embodiments, the method may further comprise detecting aeruginasin. In some embodiments, the method may further comprise detecting any two of norsyrosin, baeocystin, norbaeocystin, and aeruginasin. In some embodiments, the method may further comprise any three of norsyrosin, baeocystin, norbaeocystin, and aeruginasin. In some embodiments, the method may further comprise detecting all of norsyrosin, baeocystin, norbaeocystin, and aeruginasin.

[0068] In some embodiments, the method includes executing a mixing schedule such that, during elution of the analyte, the rate of change in the mixing ratio of solvents constituting the mobile phase of the sample during a first time period is maintained greater than the rate of change in the mixing ratio of solvents constituting the mobile phase during a second time period. During the second time period, the mixing ratio is maintained at a predetermined value. In some embodiments, during execution of the mixing schedule, at least psilocybin is separated and sequentially eluted from the column during the first time period, and at least psilocin is separated and sequentially eluted from the column during the second time period. In some embodiments, during execution of the mixing schedule, norpsilocin and aeruginasin are separated and sequentially eluted from the column during the first time period.

[0069] In some embodiments, the mixing schedule may also include a third period in which the rate of change in the mixing ratio of the solvents constituting the mobile phase is greater than the rate of change in the mixing ratio of the solvents constituting the mobile phase in the first period. In certain embodiments, when the mixing schedule including the third period is performed, baeocystin and norbaeocystin are separated and sequentially eluted from the column during the third period.

[0070] In some embodiments, the method may include generating a calibration curve for the amount of psilocybin and / or psilocin in the sample. The calibration curve may be generated using a control solution containing only psilocybin as the control analyte and / or a control solution containing only psilocin as the control analyte. Such a control solution is loaded onto the column, and the control analyte in the control solution is eluted to detect psilocybin and / or psilocin. The amount of psilocybin and / or psilocin in the control solution may be determined by comparison with known amounts of psilocybin and / or psilocin in the control solution to calibrate the method.

[0071] Therefore, by using such a method, hallucinogenic components in a sample can be detected and / or quantified. Specific examples of the present disclosure are described below. However, the examples described below are intended only to specifically illustrate and explain the present disclosure, and the scope of the present disclosure is not limited thereto. Further details regarding the implementation of the method and system disclosed herein can be further understood and explained by using the following implementation examples.

[0072] Example 1: Long Gradient, Multiple Eluents An example of LC analysis conditions used to detect hallucinogenically active ingredients in a sample, as specified in the hallucinogenic analysis control program 31 in an LC system according to the present disclosure, is as follows:

[0073] Flow rate of mobile phase: 1.0 mL / min. Gradient elution conditions in terms of the mixing ratio of mobile phase B: 1% (0 to 6 minutes) - 5% (7 minutes) - 20% (9.5 minutes) - 22% (9.6 minutes) - 22% (9.7 minutes) - 28% (9.8 minutes) - 28% (9.9 minutes) - 35% (13 minutes) - 95% (13.1 minutes) - 95% (13.6 minutes) - 1% (13.8 minutes) - 1% (17.5 minutes) - stop.

[0074] Injection volume of liquid sample: 5 μL Detection wavelength: 220 nm and / or 254 nm.

[0075] Column oven temperature: 40° C. Detector cell temperature: 40° C.

[0076] These conditions may be used to detect active ingredients such as psilocybin, psilocin, norpsilocin, baeocystin, norbaeocystin, and aeruginasin. Figure 3 shows a graph of an example chromatogram obtained from a measurement performed on a sample using the mixing schedule illustrated in the inset.

[0077] 4A-4F show graphs of the quantification of various hallucinogenic components in samples determined after implementation of the mixing schedule shown in FIG. 3 and described herein.

[0078] Example 2: Fast Gradient, Dual Eluents An example of LC analysis conditions used to detect hallucinogenically active ingredients in a sample, as specified in the hallucinogenic analysis control program 31 on an LC system according to the present disclosure, is as follows:

[0079] Flow rate of mobile phase: 1.0 mL / min. Gradient elution conditions in terms of the mixing ratio of mobile phase B: 10% (0 to 1 min) - 40% (2 min time point) - 95% (4.1 min) - 95% (4.6 min time point) - 10% (4.7 min time point) - 10% (7.4 min time point) - stop.

[0080] Injection volume of liquid sample: 5 μL Detection wavelength: 220 nm and 254 nm.

[0081] Column oven temperature: 40° C. Detector cell temperature: 40° C.

[0082] These conditions are for relatively short-term detection of active ingredients such as psilocybin and psilocin. Figure 5 shows graphs of example chromatograms obtained by measurements performed at 220 nm (top) and 254 nm (bottom) on a sample using the mixing schedule illustrated in the inset.

[0083] Although the embodiments of the present disclosure have been described, the present disclosure is not limited thereto, and various modifications can be made and executed within the scope of the claims, the detailed description of the present disclosure, and the accompanying drawings, and these also fall within the scope of the present disclosure.

Claims

1. A method for detecting psilocybin and / or psilocin in a sample, comprising: loading the sample onto a liquid chromatography column; isolating psilocybin and / or psilocin by eluting analytes in the sample; and detecting psilocybin and / or psilocin, wherein the method does not involve the use of a mass spectrometer.

2. The method of claim 1, wherein the detecting step comprises measuring the retention time of a detected peak observed in a chromatogram.

3. The method of claim 1, further comprising generating a detection peak based on the intensity and retention time measured by the detector for detecting the presence of psilocybin and / or psilocin.

4. The method of claim 1, wherein the amount of psilocybin and / or the amount of psilocin is quantified.

5. The method of claim 1 for detecting psilocybin.

6. The method of claim 1, wherein psilocin is detected.

7. The method of claim 1, wherein the analyte comprises at least one selected from the group consisting of norpsirosin, baeocystin, norbaeocystin, and aeruginasin.

8. The method of claim 1, further comprising detecting at least one species selected from the group consisting of norpsirosin, baeocystin, norbaeocystin, and aeruginasin in the sample.

9. The method of claim 1, wherein during the elution, the rate of change in the mixing ratio of the solvents constituting the mobile phase of the sample in a first period is greater than the rate of change in the mixing ratio of the solvents constituting the mobile phase in a second period, and during the second period, the mixing ratio has a predetermined constant value.

10. The method of claim 9, wherein during the first period of time, at least psilocybin is separated and sequentially eluted from the column, and during the second period of time, at least psilocin is separated and sequentially eluted from the column.

11. The method of claim 9, wherein the sample mobile phase comprises a first solution comprising water, phosphoric acid, and sodium perchlorate monohydrate.

12. The method of claim 9, wherein the sample mobile phase comprises a second solution comprising acetonitrile and phosphoric acid.

13. The method of claim 9, wherein during the first period, norsyrosin and aeruginasin are separated and eluted sequentially from the column.

14. The method of claim 9, wherein during elution, the rate of change in the mixing ratio of the solvents constituting the mobile phase in the first period is smaller than the rate of change in the mixing ratio of the solvents constituting the mobile phase in the third period, and during the third period, baeocystin and norbaeocystin are separated and eluted sequentially from the column.

15. The method of claim 1, wherein the presence or absence of each of psilocybin, psilocin, norpsilocin, baeocystin, norbaeocystin, and aeruginasin in the sample is simultaneously detected.

16. The method of claim 1, wherein the amounts of each of psilocybin, psilocin, norpsilocin, baeocystin, norbaeocystin, and aeruginasin in the sample are simultaneously detected.

17. The method of claim 1, wherein the sample is not human plasma.

18. The method of claim 1, wherein the sample is not plasma.

19. The method of claim 1, wherein the detecting step includes detecting with an ultraviolet (UV) detector.

20. The method of claim 1, wherein the detecting step includes detecting with an ultraviolet (UV) detector at a detection wavelength of 200 to 280 nm.

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