Non-invasive analysis method for liver function by gas chromatography-ion mobility spectrometry combined technology

Abstract

The invention discloses a non-invasive analysis method for liver functions by a gas chromatography-ion mobility spectrometry combined technology. The method comprises the following steps: taking limonene as a metabolic marker, fasting 4 hours before detection, orally taking lemonade or limonene capsules and rinsing with saline water 20 minutes before detection, collecting 500 [mu] L of alveolar gas, and carrying out capillary column GC separation, Fourier deconvolution IMS detection and external standard method quantification. The liver function is judged according to the limonene concentration in the range of 100 ppt-0. 1 ppm in exhaled gas, the whole process is shorter than or equal to 30 minutes, and the method is noninvasive, high in sensitivity, high in specificity and suitable for early screening and dynamic monitoring of liver diseases.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, and in particular to a non-invasive analytical method for liver function using gas chromatography-ion mobility spectrometry. Background Technology

[0002] As the body's core metabolic and detoxification organ, the liver undertakes crucial physiological functions such as substance transformation, bile secretion, and toxin removal, and its functional state is directly related to human health. Globally, over 1 billion people suffer from chronic liver disease, and many liver diseases present with insidious early symptoms. By the time typical symptoms such as jaundice and ascites appear, the disease has already progressed to the middle or late stages, significantly increasing the difficulty of treatment and medical costs. Therefore, efficient and accurate liver function analysis methods are essential for early screening of liver disease, dynamic monitoring of the disease, and evaluation of treatment effectiveness, and represent one of the core areas urgently needing breakthroughs in the field of medical testing.

[0003] Current methods for liver function analysis are mainly divided into invasive and non-invasive categories. Among invasive methods, serum biochemical marker detection (such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin) is a routine clinical procedure. It involves collecting blood from the vein to detect changes in liver metabolites. However, this method is easily affected by non-hepatic factors such as muscle injury, drug intake, and diet. It can only reflect indirect indicators of liver damage and cannot directly assess hepatocyte activity or the degree of fibrosis. Liver biopsy is considered the "gold standard" for liver function assessment. It obtains liver tissue for pathological analysis, which can accurately determine the type and extent of lesions. However, it carries the risk of complications such as bleeding, infection, and pneumothorax. Moreover, it is an invasive procedure with low patient acceptance and is not suitable for large-scale screening or long-term dynamic monitoring.

[0004] Non-invasive analytical methods have become a focus of development in recent years due to their high safety and reproducibility. These methods mainly include imaging detection, elastography, and exhaled breath testing. Imaging methods such as ultrasound, CT, and MRI can observe the size, density, and space-occupying lesions of the liver through morphological observation. However, they are not sensitive enough to early hepatocellular dysfunction or mild fibrosis. CT carries the risk of ionizing radiation, and MRI is costly and time-consuming. Transient elastography assesses the degree of fibrosis by measuring liver stiffness and is convenient to operate (about 5-10 minutes). However, its accuracy is greatly affected by factors such as patient obesity, ascites, and narrow intercostal spaces, and it cannot fully reflect liver metabolic function.

[0005] Among non-invasive analytical techniques, exhaled breath-based liver function tests exhibit unique advantages due to their complete lack of trauma and convenient sample acquisition (direct collection of exhaled breath). The technical principle is as follows: during liver metabolism, hepatocytes oxidize and decompose substances such as fatty acids and amino acids, producing volatile organic compounds (VOCs, such as methane, ethane, styrene, toluene, etc.). When liver function is impaired, abnormal metabolic pathways lead to characteristic changes in the types or concentrations of VOCs. Detecting these changes can indirectly assess liver function. Currently, this method has been applied to the auxiliary diagnosis of diseases such as alcoholic liver disease and non-alcoholic fatty liver disease. For example, the ethane concentration in the exhaled breath of patients with alcoholic hepatitis is 2-3 times higher than that of healthy individuals. However, existing exhaled breath analysis methods have significant limitations: firstly, the concentration of VOCs in exhaled breath is extremely low (mostly at the ppb or even ppt level) and is easily affected by external factors such as diet (e.g., spicy foods, alcohol), smoking, and ambient air, resulting in poor repeatability; secondly, there is a lack of specific VOCs biomarkers, making it difficult to distinguish different types of liver disease or accurately quantify the degree of liver function damage.

[0006] To address the challenge of separating and detecting VOCs in exhaled breath, gas chromatography (GC)-based analytical methods are widely used. GC efficiently separates mixed VOCs using a chromatographic column, eluting each VOC component individually based on the differences in their partition coefficients between the stationary and mobile phases. Quantitative analysis is then performed using methods such as flame ionization detectors (FID). This method boasts high separation efficiency and can effectively separate dozens or even hundreds of VOCs in exhaled breath. For example, it can accurately separate structurally similar components such as methane and ethane, or benzene and toluene, providing a foundation for screening liver function-related VOCs. However, GC analysis has inherent drawbacks: First, its qualitative ability is weak, relying solely on retention time to identify components, making it easily confused with structurally similar interfering substances and unable to accurately determine the molecular structure of VOCs; second, sample pretreatment is complex, requiring techniques such as solid-phase microextraction (SPME) to enrich VOCs in exhaled gas, which involves many steps and is time-consuming (usually more than 30 minutes), making it difficult to meet the demand for "rapid analysis"; third, the detector sensitivity is limited, with insufficient ability to detect low concentrations (<1ppb) of characteristic VOCs, affecting the early identification of abnormal liver function.

[0007] To overcome the limitations of GC in qualitative analysis, gas chromatography-mass spectrometry (GC-MS) has been developed. This method combines the high separation efficiency of GC with the high sensitivity and specificity of MS. VOCs separated by GC are introduced into an MS detector, ionized, and generate characteristic mass spectra. By comparing these spectra with a standard library, the structure of the components can be accurately determined, and trace concentrations can be detected simultaneously. For example, studies have found that the concentration of styrene in the exhaled breath of patients with cirrhosis is 2-3 times higher than that in healthy individuals. GC-MS can accurately identify and quantify this component through characteristic ion peaks (e.g., m / z=104), providing a specific biomarker for liver function assessment. However, the GC-MS method still has significant limitations: First, the instrument is large and expensive, requiring professional personnel for operation and maintenance, making it impossible to achieve rapid on-site testing or widespread use in primary healthcare institutions; second, the analysis cycle is long, taking 1-2 hours from sample pretreatment and GC separation to MS detection, which is difficult to meet the "rapid" requirements of emergency or large-scale screening scenarios; third, contamination is easily introduced during sample pretreatment, affecting the accuracy of detection, and it has poor tolerance to high humidity exhaled gases, requiring additional dehydration treatment, further increasing the complexity of operation.

[0008] In summary, existing liver function analysis methods all have significant drawbacks and limitations: invasive methods are highly traumatic, have low patient acceptance, and are unsuitable for routine screening; non-invasive imaging methods lack sensitivity to early functional abnormalities and involve radiation or cost issues; exhaled gas-based analysis methods, while completely non-invasive, are susceptible to numerous interference factors and have limited detection accuracy; GC and GC-MS methods, although improving VOCs separation and detection capabilities, suffer from expensive equipment, complex operation, and lengthy analysis times, failing to meet the clinical needs of being "non-invasive, rapid, convenient, and accurate." Therefore, developing a liver function analysis method that balances non-invasiveness, speed, high accuracy, and portability has become a key direction for overcoming current technological bottlenecks. Summary of the Invention

[0009] The purpose of this invention is to address the shortcomings of existing technologies by providing a non-invasive analytical method for liver function using gas chromatography-ion mobility spectrometry (GC-IMS). This method solves the problems of invasive methods being highly invasive, and non-invasive methods having insufficient sensitivity, complex operation, high cost, long analysis cycles, low concentration of volatile organic compounds, numerous interfering factors, and difficulty in detecting specific biomarkers in exhaled gas analysis.

[0010] This invention provides a non-invasive and rapid liver function analysis technique based on gas chromatography-ion mobility spectrometry (GC-IMS). Using limonene as a specific metabolic biomarker, this technique optimizes sample pretreatment, sampling methods, and separation and detection parameters to achieve efficient end-to-end detection from direct exhaled breath sampling to accurate interpretation of liver function results. It combines non-invasiveness, speed, high sensitivity, and high specificity, meeting the needs of early clinical screening and dynamic monitoring.

[0011] This invention provides a core principle based on the correlation between the liver's metabolic capacity for specific exogenous substances and its functional state: Limonene, as a monoterpene compound, is primarily metabolized by the liver's cytochrome P450 enzyme system (especially CYP2C9 and CYP3A4) after entering the human body, with the metabolites excreted through urine or pulmonary gas exchange. When liver function is impaired (e.g., hepatocellular necrosis, fibrosis, cirrhosis), the activity of liver metabolic enzymes decreases, slowing the metabolic rate of limonene. This leads to an increase in the concentration of unmetabolized limonene in the blood, which then crosses the alveolar air-blood barrier into exhaled air, resulting in a significantly higher residual amount of limonene in exhaled air compared to healthy individuals. Therefore, by detecting the residual amount of limonene in exhaled air, liver metabolic function can be indirectly quantified, enabling the assessment of liver function status.

[0012] Meanwhile, GC-IMS technology combines the efficient component separation capability of gas chromatography with the high sensitivity and rapid response of ion mobility spectrometry: gas chromatography can efficiently separate complex VOC mixtures (including limonene and interfering components) in exhaled breath, avoiding matrix interference; ion mobility spectrometry can perform trace detection of the separated limonene. In particular, after optimization by Fourier deconvolution multiplexing technology, the detection resolution and sensitivity can be further improved, meeting the precise quantitative requirements of limonene at the ppb level or even ppt level, and solving the problem of insufficient detection capability of traditional detection methods for low-concentration markers.

[0013] The detailed technical solution of this invention is as follows: (a) Sample pretreatment and biomarker introduction To eliminate external interference and ensure the specificity and repeatability of limonene detection, this invention establishes a strict sample pretreatment process, the specific steps of which are as follows: Fasting and Basal Status Control: For 4 hours prior to the test, the subject must strictly fast (a moderate amount of pure water may be consumed, but alcoholic, spicy, and aromatic foods and beverages are prohibited). This step prevents VOCs in food (such as terpenoids in spices and alcohol metabolites) from entering the exhaled breath, reducing matrix interference for limonene detection, while ensuring the subject is in a stable metabolic basal state and avoiding fluctuations in liver metabolic enzyme activity caused by diet.

[0014] Limonene introduction method: 20 minutes before the test, limonene is introduced using two standardized methods to ensure that the concentration of limonene in the subject's body is within a controllable range, and the two methods can be flexibly selected according to the clinical scenario: Method 1: Oral administration of lemon water. The lemon water should be prepared according to a standardized procedure: take D-limonene standard with a purity of ≥99.5%, dilute it with pure water to a concentration of 0.5mg / mL, add 0.1g of food-grade citric acid to every 100mL of dilution to adjust the taste (avoid pH values ​​that are too high or too low and irritate the oral mucosa), and the subject should take 200mL of the lemon water orally at one time.

[0015] Method 2: Take limonene capsules. Each capsule contains 50mg of D-limonene with a purity of ≥99.5%. The capsule shell is made of hydroxypropyl methylcellulose (to avoid the generation of interfering substances due to capsule shell degradation). The subject should take one capsule with 100mL of pure water.

[0016] Oral residue removal: After limonene introduction, the subject needs to rinse their mouth three times with 0.9% saline solution, each time for ≥30 seconds, and spit out the saline solution after rinsing. This step can remove any unswallowed limonene residue in the mouth, preventing it from directly entering the sampling gas and causing an overestimation of the test results. This ensures that the limonene ultimately detected originates only from pulmonary gas exchange (i.e., the residual amount that enters the bloodstream after being metabolized by the liver).

[0017] (II) Exhaled gas sampling method After pretreatment (i.e., 20 minutes after the introduction of limonene), alveolar gases were collected using two standardized sampling methods to ensure accurate sampling volume and strong gas representativeness, as detailed below: Direct syringe sampling method: Use a 1mL airtight glass syringe (the syringe inner wall is silanized to prevent limonene adsorption). Have the subject take three normal breaths, followed by a fourth deep breath and 5 seconds of breath-holding (to ensure sufficient gas exchange to the alveoli). Then, slowly exhale the gas into the syringe. Stop sampling when the syringe reaches 1mL and immediately seal the syringe port with a rubber cap. After sampling, injection must be completed within 5 minutes to prevent limonene adsorption or diffusion within the syringe, which could cause concentration changes. During injection, connect the syringe needle to the gas chromatograph's inlet and slowly push the plunger to inject 500μL of alveolar gas (injection rate controlled at 0.1mL / s to avoid peak broadening due to gas flow impact).

[0018] Six-way valve-inlet sampling method: A six-way valve with a quantitative loop (500 μL volume) is used as the sampling and injection switching device. One end of the six-way valve is connected to a dedicated inlet (the inner wall of the inlet is made of polytetrafluoroethylene to reduce adsorption), and the other end is connected to the gas chromatograph injection port. During sampling, the subject holds the inlet in their mouth, takes a deep breath, holds their breath for 5 seconds, and slowly blows air into the inlet. The airflow passes through the six-way valve into the quantitative loop. When the quantitative loop is full (the airflow volume reaches 500 μL as monitored by a flow meter), the six-way valve is closed. During injection, the six-way valve path is switched, and the carrier gas delivers the 500 μL of alveolar gas in the quantitative loop into the gas chromatograph for separation. This method avoids human error during syringe sampling, further improving sampling repeatability, and the RSD (relative standard deviation) can be controlled within 3%.

[0019] (III) Parameters of Gas Chromatography Separation System This invention employs temperature-programmed gas chromatography to achieve efficient separation of limonene and interfering components in exhaled breath. The core parameters are as follows: Column selection: DB-35 capillary column (size: 30m×0.25mm×0.25μm) was selected. The stationary phase of this column is 35% phenyl-65% dimethyl polysiloxane, which has good separation ability for both polar and non-polar VOCs. In particular, the resolution of limonene (a non-polar compound) and common interfering substances in exhaled gas (such as ethanol, acetic acid, styrene, etc.) is ≥2.0, which can effectively avoid the influence of interfering peaks on the detection of limonene.

[0020] Carrier gas and flow rate control: High-purity nitrogen (purity ≥99.999%) was used as the carrier gas, and a constant flow mode was adopted with the column flow rate set at 1.0 mL / min. This flow rate ensures sufficient separation of limonene within the column while controlling the separation time to avoid excessively long analysis cycles.

[0021] The programmed temperature ramp is as follows: the initial temperature is set at 40°C and held for 1 minute (to separate low-boiling-point interfering substances, such as ethanol and water vapor); then the temperature is increased to 200°C at a rate of 12°C / min and held for 1 minute (to remove high-boiling-point residues from the column and avoid cross-contamination). The entire chromatographic separation process takes 10 minutes, which is much shorter than traditional GC-MS methods, meeting the requirements for rapid, high-throughput analysis.

[0022] Injector parameters: The injection port temperature is set to 120℃, and a split injection mode with a split ratio of 10:1 is used. Split injection can reduce peak broadening caused by excessive injection volume, while avoiding contamination of the column by high concentrations of interfering substances, ensuring stable separation efficiency.

[0023] (iv) Parameters of the ion mobility spectrometry detection system This invention uses ion mobility spectrometry as a detector. Through optimized detection modes and technological improvements, it achieves trace detection and precise qualitative and quantitative analysis of limonene. The core parameters are as follows: Detection mode and ion source: The ion mobility spectrum adopts the positive ion mode and the ion source is the nickel-63 source. This ion source has the advantages of high stability, long life (half-life of about 100 years) and no need for an external power supply. It can ionize limonene molecules into the characteristic ion [limonene + H] +, avoiding the problem of being easily interfered with by water vapor in the negative ion mode.

[0024] Migration tube parameters: The migration tube temperature was set to 80℃, the migration zone length was 10cm, high-purity nitrogen (purity ≥99.999%) was used as the drift gas, and the drift gas flow rate was set to 150mL / min. These temperature and flow rate parameters ensured the stable migration of [limonene + H]+ ions within the migration tube, avoiding migration time deviations caused by temperature fluctuations. The migration time of the limonene characteristic ion was stabilized at 9.2±0.1ms.

[0025] High resolution and high sensitivity optimization: The preferred Fourier deconvolution multiplexed ion mobility spectrometry technique is used. By applying a periodically changing electric field in the migration tube, different ions generate multiplexed signals. The signals are then processed by the Fourier deconvolution algorithm to separate overlapping ion migration peaks, improving the resolution by 1 times compared to traditional IMS (it can distinguish ions with a migration time difference ≤ 0.05 ms). At the same time, this technique reduces the limit of detection (LOD) of limonene to 20 ppt through the signal accumulation effect, which is much lower than that of traditional IMS (LOD of about 1 ppt), meeting the detection requirements in the linear range of 100 ppt-0.1 ppm.

[0026] Qualitative and quantitative methods: Qualitative analysis: A dual-dimensional qualitative approach using retention time and migration time was employed. After separation by gas chromatography, the retention time of limonene was fixed at 7.65 ± 0.02 minutes; after entering ion mobility spectrometry, the migration time of its characteristic ions was fixed at 9.2 ± 0.1 ms. Combining these two methods allows for precise differentiation of limonene from structurally similar interfering substances (such as α-pinene and β-pinene, whose retention and migration times differ significantly from limonene), with a qualitative accuracy ≥ 99%.

[0027] Quantitative analysis: An external standard method was used to establish a standard curve using limonene standard solution. The standard curve preparation process is as follows: D-limonene standard with a purity ≥99.5% was diluted with nitrogen to standard gas concentrations of 0.1 ppb (100 ppt), 1 ppb, 10 ppb, 20 ppb, 50 ppb, and 100 ppb (0.1 ppm) (a total of 5 concentration points). Each concentration point was injected three times. The peak area of ​​limonene (integrated value of ion mobility spectrum response signal) was used as the ordinate and the concentration as the abscissa. A linear regression equation was used to fit the curve, and the standard curve equation was obtained as y=0.0406x+0.3884 (R²≥0.9981). The linear range covers 100 ppt-0.1 ppm, which meets the detection requirements of limonene residue under different liver function states (the concentration of limonene in the exhaled breath of healthy people is about 100-300 ppt, about 300 ppt-0.05 ppm in people with mild liver damage, and ≥0.05 ppm in people with moderate to severe liver damage).

[0028] The present invention provides a non-invasive analytical method for liver function using gas chromatography-ion mobility spectrometry, which has the following beneficial effects: Completely non-invasive: The entire process only collects the subject's exhaled breath, without the need for blood collection or puncture. Patient acceptance is high. Clinical surveys show that more than 95% of subjects prefer this method to serum testing. It can be used for large-scale screening or long-term dynamic monitoring, such as tracking the treatment effect of patients with chronic liver disease.

[0029] Fast and efficient: The entire process from sampling to result interpretation takes ≤30 minutes (20 minutes for preprocessing + ≤10 minutes for separation and detection), which is much shorter than traditional GC-MS (1-2 hours) and MRI (30-60 minutes). It can meet the rapid detection needs of emergency scenarios, such as the emergency assessment of drug-induced liver injury.

[0030] High sensitivity and specificity: Based on Fourier deconvolution multiplexing IMS technology, the detection limit of limonene is as low as 50 ppt, and the linear range covers 100 ppt-0.1 ppm, which can accurately detect changes in limonene concentration in early liver injury; the dual qualitative mode of "retention time + migration time" avoids interference and achieves a specificity of 90.5%.

[0031] Easy to operate and cost-controllable: The GC-IMS combined instrument is small in size and low in cost, requires no professional operators, and can be widely used in primary medical institutions.

[0032] Strong anti-interference ability: Through a strict pre-processing procedure and efficient GC separation, it effectively eliminates interference factors such as food, oral residue, and ambient air. The repeatability RSD is ≤5% (n=10), which is far superior to traditional exhaled gas detection methods (RSD about 10-15%). Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the system structure according to an embodiment of the present invention; Figure 2 (a) is a two-dimensional gas chromatography-ion mobility spectrometry (GC-IMS) spectrum of the characteristic peaks of limonene, (b) is a chromatogram of D-limonene, and (c) is an ion mobility spectrum of D-limonene. Figure 3 This is the quantitative curve of the characteristic peaks of limonene; Figure 4 This is a two-dimensional gas chromatography-ion mobility spectrometry (GC-IMS) spectrum showing the characteristic peak of limonene detected in exhaled breath.

[0034] Illustration: 1-Ion source; 2-Reaction zone; 3-Ion gate; 4-Migration zone; 5-Faraday disk; 6-Gas chromatography module. Detailed Implementation

[0035] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. It should be noted that the following embodiments should not be construed as limiting the scope of protection of the present invention. If those skilled in the art make some non-essential improvements and adjustments to the present invention based on the above description, they shall still fall within the scope of protection of the present invention. Example

[0036] This embodiment provides a non-invasive and rapid detection method for liver function assessment. By standardizing the introduction of limonene as a liver metabolic probe, and using highly sensitive GC-IMS technology to detect the concentration of residual limonene in exhaled gas, the metabolic function of the liver can be indirectly assessed.

[0037] 1. GC-IMS device (1) The overall GC-IMS device structure used in this invention is as follows: Figure 1 As shown, the system consists of a gas chromatography (GC) module and an ion mobility spectrometry (IMS) detector coupled online.

[0038] (2) Gas Chromatography Module 6: Its core function is to perform efficient and rapid separation of complex exhaled gas samples. The sample first enters this module, and by utilizing the difference in the partition coefficients of different components between the stationary phase and the mobile phase of the chromatographic column, separation is achieved in the time dimension, laying the foundation for subsequent accurate detection.

[0039] Ion mobility spectrometry (IMS) detector: As the core detection unit, it includes the following components sequentially along the ion transport path: Ion Source 1: This invention preferably uses a radioactive nickel-63 source. This ion source has advantages such as high stability, long lifetime (half-life of approximately 100 years), and no need for an additional complex power supply, and can continuously generate uniform reaction ions. In positive ion mode, the reaction ions undergo a proton transfer reaction with limonene molecules that have been separated by GC, efficiently and stably ionizing them into the characteristic ion [limonene + H]⁺. Choosing positive ion mode can effectively avoid the suppression and interference of a large amount of water vapor in exhaled gas on the ionization process and ion signal in negative ion mode.

[0040] Reaction Zone 2: In this zone, the ionized sample ions fully interact with the reaction ions and reach stability.

[0041] Ion gate 3: An electrically controlled gate is used, which opens periodically with extremely narrow pulses to cut the continuous ion flow into discrete ion pulse packets with time start points, which are then injected into the migration region.

[0042] Migration Zone 4: A constant weak electric field is applied in an atmospheric pressure tube filled with a uniform reverse drift gas (high-purity nitrogen). Ions with different mass-to-charge ratios and collision cross-sections experience different electric forces and drift gas resistance, thus migrating at different speeds and achieving secondary separation in the spatial dimension.

[0043] Faraday disk 5: Ions reaching the end of the migration region are collected by this Faraday disk and converted into electrical signals, which are then amplified and recorded to form an ion migration spectrum.

[0044] (3) The GC module and the IMS detector are connected to the gas chromatography module 6 through a temperature-controlled transfer line to ensure that the components separated by GC can directly and without condensation enter the IMS for ionization and detection.

[0045] 2. Qualitative analysis and system parameter optimization of limonene (1) First, qualitative analysis of limonene standards was performed to establish the basis for detection. High-purity nitrogen was introduced into the dilution container as a dilution gas. A certain amount of D-limonene standard with a purity ≥99.5% was dissolved in dichloromethane and added to the dilution container. After evaporation, a standard gas with a concentration of 500 ppt was prepared. 500 μL of the standard gas was drawn using a 1 mL airtight glass syringe (the inner walls of the dilution container and the syringe were silanized to minimize the adsorption of limonene). The syringe needle was connected to the injection port of the gas chromatograph and injected.

[0046] (2) The peak positions of limonene characteristic ions were determined by GC-IMS system detection. For example... Figure 2As shown, in the optimized system of this invention, the retention time of the characteristic ion peak of limonene is fixed at 7.65±0.02 min, and the migration time is fixed at 9.20±0.01 ms. This invention uses this dual-dimensional information of "retention time + migration time" to accurately characterize the analyte, and can effectively distinguish structurally similar interfering substances.

[0047] (3) Based on this, the key parameters of gas chromatography (GC) and ion mobility spectrometry (IMS) were systematically optimized, and the final experimental parameters were as follows: Gas chromatography (GC) parameters: DB-35 capillary column (30m×0.25mm×0.25μm) was used. High-purity nitrogen (≥99.999%) was used as the carrier gas, constant flow mode, and the column flow rate was 1.0mL / min. The initial temperature of the programmed temperature rise was set to 40℃ and held for 1 minute; then the temperature was increased to 200℃ at a rate of 12℃ / min and held for 1 minute. The injection port temperature was 120℃ and the injection split ratio was 10:1. Ion mobility spectrometry (IMS) parameters: The migration tube temperature was set to 80℃, the migration zone length was 10cm, high-purity nitrogen (purity ≥99.999%) was used as the drift gas, and the drift gas flow rate was set to 150mL / min.

[0048] 3. Quantitative methods, precision, and stability studies (1) Quantitative method: The external standard method is used for quantification in this invention. Standard gases with concentrations of 0.1 ppb (100 ppt), 1 ppb, 10 ppb, 20 ppb, 50 ppb, and 100 ppb (0.1 ppm) were prepared using D-limonene standards. Each concentration point was injected three times, and a linear regression was performed on the concentration using the peak area. Figure 3 As shown, the standard curve equation is obtained as y = 0.0406x + 0.3884 (R² ≥ 0.9981), with a linear range covering 100 ppt to 0.1 ppm.

[0049] (2) Precision verification: To examine the repeatability (precision) of the method, limonene standard gas at concentrations of 200 ppt and 0.05 ppm were injected six times each, and the relative standard deviation (RSD) of the peak area was calculated. The results showed that the peak area RSD of the 200 ppt concentration group was 2.3%, and the peak area RSD of the 0.05 ppm concentration group was 1.8%.

[0050] (3) Stability Verification: To examine the long-term stability of the instrument and method, the same instrument was run continuously for 3 days, and the peak area and migration time of limonene standard gas at a concentration of 500 ppt were detected daily. The results showed that the RSD of the peak area was 3.5% and the RSD of the migration time was 0.8% over the 3 consecutive days. This data indicates that both the method and the instrument system exhibit good long-term stability in terms of both the intensity of the detection signal (peak area) and the stability of the qualitative parameters (migration time), meeting the requirements for long-term continuous use.

[0051] 4. Actual exhaled breath sample testing and method validation (1) Actual Sample Detection Method: To verify the effectiveness of this method in practical applications, subjects were recruited for actual exhaled breath testing. The specific procedure is as follows: Subjects were strictly fasting for 4 hours before the test (only pure water was allowed). Subsequently, subjects orally ingested 200 mL of lemon water (containing 0.5 mg / mL of D-limonene) prepared according to the standard of this invention. 20 minutes after the introduction of the marker, alveolar air was collected using the direct sampling method with a syringe.

[0052] (a) The specific operational details of the direct syringe sampling method are as follows: Preparation before sampling: Use a 1 mL airtight glass syringe with its inner wall pre-treated with silanization inertness to minimize adsorption loss of the target analyte (limonene) on the syringe wall. Alveolar gas collection: Instruct the subject to sit calmly and take 2-3 normal breaths. Then, instruct them to take a deep breath to their lung capacity and hold it for 5 seconds. This step aims to ensure sufficient gas exchange between the inhaled gas and the alveolar blood, and to allow limonene to reach equilibrium in the alveolar gas. After holding their breath, the subject should quickly remove their lips, immediately align the syringe exhaust port with their mouth, and then slowly and continuously exhale the alveolar gas. The operator should observe the syringe scale and stop sampling immediately when the gas volume reaches 1.0 mL. Sample sealing and preservation: After sampling, immediately seal the syringe port tightly with a pre-prepared rubber sealing cap to prevent sample leakage or mixing with ambient air. The entire process from sampling to GC-IMS injection is strictly controlled within 5 minutes to avoid concentration changes that may occur due to prolonged sample residence in the syringe.

[0053] (b) Standardized injection: During injection, insert the syringe needle vertically into the injection septum of the gas chromatograph. Push the piston at a uniform and slow speed to accurately inject 500 μL of gas sample, with the injection rate strictly controlled at 0.1 mL / s. This low-speed injection mode helps avoid peak broadening caused by gas flow impact, ensuring sharp and symmetrical peak shapes.

[0054] (c) Immediately after sample injection, perform GC-IMS analysis. The experimental results are as follows: Figure 4As shown. By substituting the peak intensity (peak area) of limonene detected in actual exhaled breath into the standard curve established by this standard method, the accurate content of limonene in exhaled breath can be calculated.

[0055] (2) Recovery Experiment: To verify the accuracy of quantitative analysis, a spiked recovery experiment was conducted. Three simulated exhaled gas samples of known concentrations (nitrogen matrix, containing 150 ppt, 0.04 ppm, and 0.09 ppm limonene, respectively) were taken and analyzed using this method. Each concentration level was measured in triplicate. The measured values ​​were compared with the known concentrations to calculate the recovery rate. The results showed that the average recovery rate for the three concentration levels was between 90.2% and 108.5%. This recovery rate range fully meets the accuracy requirements for trace analysis, proving that the quantitative results of this method are accurate and reliable.

[0056] (3) Detection and application of different actual samples: This method was applied to healthy volunteers and patients with different degrees of liver injury. As a blank control, the same subject was sampled after ingesting 200 mL of high-purity water on different test days. No obvious limonene characteristic peaks were detected in the spectral data, proving the effectiveness of the pretreatment process. The results of the detection on the actual population showed that the concentration of limonene in the exhaled breath of healthy people was about 100-300 ppt; the concentration in patients with mild liver injury was about 300 ppt-0.05 ppm; while the concentration in patients with moderate to severe liver injury was generally ≥0.05 ppm. This result is consistent with the pathophysiological mechanism of impaired liver metabolism leading to slowed limonene clearance, fully demonstrating that the method of the present invention can effectively distinguish different liver function states and has significant clinical application value.

[0057] The embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention.

Claims

1. A non-invasive analytical method for liver function using gas chromatography-ion mobility spectrometry, characterized in that, include: Step 1: The subject should fast for 4 hours before the test and take lemon water or limonene capsules orally 20 minutes before the test. After taking the capsules, rinse your mouth with saline solution to remove any unswallowed limonene residue. Step 2: Alveolar air was collected using two sampling methods: direct syringe sampling and six-way valve-inlet sampling. Step 3: Separate limonene from interfering components in exhaled breath using temperature-programmed gas chromatography. Step four: Ion mobility spectrometry was used as a detector to qualitatively identify limonene using a two-dimensional method of retention time and migration time; an external standard method was used to establish a standard curve with limonene standard solutions. Step 5: Assess liver function based on the limonene content in exhaled breath.

2. The non-invasive analytical method for liver function using gas chromatography-ion mobility spectrometry as described in claim 1, characterized in that, In step one, the lemon water is prepared using a standardized method: D-limonene standard with a purity of ≥99.5% is taken and diluted with pure water to a concentration of 0.5 mg / mL. 0.1 g of food-grade citric acid is added to every 100 mL of diluted solution to adjust the taste. The test subject takes 200 mL of the lemon water orally at one time.

3. The non-invasive analytical method for liver function using gas chromatography-ion mobility spectrometry as described in claim 1, characterized in that, In step one, the limonene capsules are specified to contain 50mg of D-limonene with a purity of ≥99.5% per capsule. The capsule shell is made of hydroxypropyl methylcellulose. The subject takes one capsule with 100mL of pure water.

4. The non-invasive analytical method for liver function using gas chromatography-ion mobility spectrometry as described in claim 1, characterized in that, In step one, after the limonene is introduced, the subject rinses his mouth three times with 0.9% saline solution, each time for ≥30 seconds, and then spits out the saline solution.

5. The non-invasive analytical method for liver function using gas chromatography-ion mobility spectrometry as described in claim 1, characterized in that, In step two, the direct syringe sampling method includes: Use a 1mL airtight glass syringe with a silanized inner wall. Instruct the subject to take three normal breaths, hold their breath for five seconds after the fourth deep breath, and then exhale into the syringe. Stop sampling when the syringe reaches 1mL and seal the syringe port with a rubber cap. After sampling, the sample must be injected within 5 minutes. During injection, connect the syringe needle to the gas chromatograph's inlet, push the piston, and inject 500μL of alveolar gas at a rate of 0.1mL / s.

6. The non-invasive analytical method for liver function using gas chromatography-ion mobility spectrometry as described in claim 1, characterized in that, In step two, the six-way valve-blowing inlet sampling method includes: A six-way valve with a quantitative loop and a volume of 500 μL is used as the sampling and injection switching device. One end of the six-way valve is connected to a dedicated inlet, the inner wall of which is made of polytetrafluoroethylene (PTFE), and the other end is connected to the gas chromatograph injection port. During sampling, the subject holds the inlet in their mouth, takes a deep breath, holds their breath for 5 seconds, and blows air into the inlet. The airflow enters the quantitative loop through the six-way valve. When the quantitative loop is full, the flow rate is monitored by a flow meter to reach 500 μL, and then the six-way valve is closed. During injection, the six-way valve path is switched, and the carrier gas delivers the 500 μL of alveolar gas in the quantitative loop into the gas chromatograph for separation.

7. The non-invasive analytical method for liver function using gas chromatography-ion mobility spectrometry as described in claim 1, characterized in that, In step three, gas chromatography separation was performed using a DB-35 capillary column with 99.999% high-purity nitrogen as the carrier gas. The column flow rate was 1.0 mL / min, and the temperature program was as follows: 40℃ for 3 min, 5℃ / min to 120℃ for 2 min, 20℃ / min to 200℃ for 1 min, and the injection port temperature was 200℃ with a split ratio of 10:

1.

8. The non-invasive analytical method for liver function using gas chromatography-ion mobility spectrometry as described in claim 1, characterized in that, In step four, ion mobility spectrometry detection was performed in positive ion mode, with a nickel-63 ion source, a migration tube temperature of 80℃ and a length of 10cm, and a drift gas of 99.999% high-purity nitrogen at a flow rate of 150mL / min. Fourier deconvolution multiplexing technology was used to qualitatively analyze limonene in two dimensions: retention time and migration time.

9. The non-invasive analytical method for liver function using gas chromatography-ion mobility spectrometry as described in claim 1, characterized in that, In step four, the external standard method was used for quantification. A standard curve with a linear range of 100 ppt to 0.1 ppm was established using limonene standard solution. After fitting the standard curve equation, R² ≥ 0.998 and the limit of detection ≤ 50 ppt.

10. The non-invasive analytical method for liver function using gas chromatography-ion mobility spectrometry as described in claim 1, characterized in that, In step five, the following liver function assessment criteria were used: limonene concentration of 100-300 ppt was considered healthy, 300 ppt-0.05 ppm was considered mild liver damage, 0.05-0.08 ppm was considered moderate liver damage, and ≥0.08 ppm was considered severe liver damage.

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