Breathing sampler and exhaled gas detection method

Abstract

The embodiment of the invention discloses a respiration sampler and an exhaled gas detection method. The breathing sampler comprises a gas inflow channel, a gas collection bottle and a gas outflow channel which are sequentially connected, one-way valves are arranged on the gas inflow channel and the gas outflow channel respectively, and the gas collection bottle is provided with a sealing cover and internally contains an extraction solvent. The outlet end of the gas inflow channel and the inlet end of the gas outflow channel are both located in the gas collection bottle, the outlet end of the gas inflow channel is located below the liquid level of the extraction solvent, and the inlet end of the gas outflow channel is located above the liquid level of the extraction solvent. The invention provides a valuable insight for the breathing PPA of HPA patients, showing the possibility of the breathing sampler as a potential tool for non-invasive monitoring and evaluation of HPA, making it possible to perform HPA evaluation and monitoring through non-invasive breathing samples, but non-invasive blood samples, and being particularly suitable for convenient family sampling.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of detection technologies, and particularly to a breath sampler and an exhaled gas detection method. Background Art

[0002] Hyperphenylalaninemia (HPA) is the most common congenital amino acid metabolism-related defect. It is a genetic metabolic disease caused by a deficiency of phenylalanine hydroxylase or tetrahydrobiopterin, an enzyme responsible for converting phenylalanine (Phe) into tyrosine. Untreated HPA can lead to intellectual disability, seizures, behavioral problems, and mental disorders. Although there is no definite and effective treatment for HPA, it is one of the few genetic diseases that can be controlled by dietary or drug intervention. Therefore, correct monitoring and treatment of HPA help prevent limitations in the behavior and health problems of major HPA patients. There is an urgent need for a rapid and accurate HPA detection method in clinical applications and health management.

[0003] To date, various analytical tools have been developed for detecting HPA. Most countries usually include HPA in the neonatal screening panel and use various detection techniques. Most infants are screened for HPA a few days after birth using dried blood spot (DBS) samples collected by neonatal heel prick. HPA screening has been performed by bacterial inhibition tests, biosensors, immunoassays using fluorescence or photometric detection, or amino acid assays using chromatography. Mass spectrometry (MS) is one of the most powerful analytical techniques for characterizing various analytes in clinical applications due to its high sensitivity and specificity. Tandem mass spectrometry (MS / MS) is usually performed for blood spot analysis using electrospray ionization, atmospheric pressure chemical ionization, and ambient ionization techniques. These blood detection methods can provide accurate test results for HPA diagnosis; however, the invasiveness of blood tests limits frequent blood sampling. Currently, metabolic analysis of exhaled breath has attracted great attention in the field of disease diagnosis and research. In HPA patients, due to abnormal metabolism of phenylalanine (Phe), its metabolite phenylpyruvic acid (PPA) accumulates in large amounts, usually resulting in a characteristic odor in the sweat, breath, skin, and urine of patients. Therefore, breath analysis can provide a non-invasive clinical detection approach for the rapid assessment and monitoring of HPA. Mass spectrometry (MS)-based breath analysis has been proven to be a powerful analytical strategy in clinical applications such as disease research, diagnosis, and monitoring. Currently, a variety of MS-based breath sampling and analysis techniques have been successfully applied to the field of breath analysis. Direct MS methods can achieve on-line analysis of exhaled breath samples. However, due to the extremely low concentration of ultra-trace analytes present in exhaled breath samples, it poses a challenge in the detection of exhaled breath samples. To this end, a variety of breath samplers have been developed for capturing breath metabolites, including gas containers (e.g., air bags, bottles), adsorption devices (e.g., adsorbents in fibers, membranes, strips, tubes), and condensation devices, etc. However, these exhaled breath samplers focus on collecting volatile breath metabolites, and non-volatile metabolites usually require condensation enrichment or chemical derivatization, which is time-consuming and cumbersome in pretreatment. Therefore, it remains a challenging task to capture non-volatile metabolites using a non-condensing sampler at room temperature. Summary of the Invention

[0004] To this end, an embodiment of the present invention provides a breath sampler and an exhaled gas detection method to solve the problems of low sampling efficiency of non-volatile metabolites (such as PPA) and cumbersome pretreatment in the prior art, resulting in insufficient detection sensitivity and poor operation timeliness.

[0005] To achieve the above object, the embodiment of the present invention provides the following technical solutions:

[0006] According to the first aspect of the embodiments of the present invention, the present invention provides a breath sampler, which includes a gas inlet channel, a gas collecting bottle, and a gas outlet channel connected in sequence. One-way valves are respectively arranged on the gas inlet channel and the gas outlet channel. The gas collecting bottle is equipped with a sealing cover and contains an extraction solvent inside. The outlet end of the gas inlet channel and the inlet end of the gas outlet channel are both located inside the gas collecting bottle, and the outlet end of the gas inlet channel is below the liquid level of the extraction solvent, while the inlet end of the gas outlet channel is above the liquid level of the extraction solvent.

[0007] Further, an interface adapted to the shape of the human mouth and nose is provided at the inlet end of the gas inlet channel.

[0008] Further, a flow valve is provided on the gas outlet channel.

[0009] Further, the extraction solvent is ethanol.

[0010] Further, the gas inlet channel, the gas collecting bottle, and the gas outlet channel are connected by a detachable connection method.

[0011] According to the second aspect of the embodiments of the present invention, the present invention provides an exhaled gas detection method based on the breath sampler described in any one of the above. The method includes:

[0012] Blow out exhaled gas from the inlet end of the gas inlet channel and collect the exhaled liquid in the gas collecting bottle;

[0013] Use an LC-MS / MS instrument to detect the exhaled liquid to obtain the PPA concentration of the exhaled gas.

[0014] Further, after taking a deep breath for 5 s, blow out exhaled gas at a flow rate of 0.2 - 0.3 L / min, and the number of times of blowing out exhaled gas is 6 times.

[0015] Further, the exhalation time is 1 minute.

[0016] Further, the voltage of ESI is 4.5 kV, the temperature of ESI is 550 °C, the nebulizing gas is 70 psi, the auxiliary heating is 10 L / min, the curtain gas is 3.0 mL / min, and the collision gas is 0.3 mL / min.

[0017] The embodiments of the present invention have the following advantages:

[0018] Despite efforts to prevent hyperphenylalaninemia (HPA) during prenatal examinations, many infants with HPA are still found in neonatal screening. Since there is no clear and effective treatment method for HPA, routine examinations and monitoring are crucial for effectively managing the HPA condition through dietary restrictions.

[0019] Blood phenylalanine (Phe) is one of the metabolic biomarkers of HPA and is thus considered an indicator for HPA management, but there are challenges in invasive sampling. Phenylpyruvic acid (PPA) is also a biomarker of HPA and is significantly elevated in the blood and urine of patients.

[0020] The present invention developed a breath sampler with a simple structure, which includes a tube and an extraction solvent for in-situ extraction and storage of PPA in the exhaled metabolites of HPA patients and healthy volunteers, and is coupled with mass spectrometry (MS) to achieve on-site sampling and storage of breath samples. The respiratory metabolites are extracted by pre-depositing an organic solvent in the sampling tube, and the sampling conditions for exhaled PPA detection are optimized.

[0021] High-resolution MS / MS and triple quadrupole MS / MS were used in the study to detect the breath samples of HPA patients and healthy volunteers respectively, identify and quantify exhaled PPA and blood Phe, and compare the concentrations of respiratory PPA and blood Phe. The results showed that there were significant differences in respiratory PPA between HPA patients and healthy volunteers, and there was a linear correlation between respiratory PPA and blood Phe, indicating that respiratory PPA could potentially reflect blood Phe levels.

[0022] Overall, this work provides valuable insights into respiratory PPA in HPA patients, showing the possibility of this breath sampler as a potential tool for non-invasive monitoring and assessment of HPA, making it possible to perform HPA evaluation and monitoring through non-invasive breath samples instead of invasive blood samples, especially suitable for convenient home sampling. Description of the Drawings

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, without creative efforts, other implementation drawings can be obtained according to the provided drawings.

[0024] Figure 1 Schematic structural diagram of the exhalation sampler provided by the present invention;

[0025] Figure 2 Identification of respiratory PPA provided by the present invention, wherein, a. Detection of PPA in nitrogen flow; b. Detection of respiratory PPA in HPA patients;

[0026] Figure 3 Optimization of PPA sampling in the gas flow of the portable injector provided by the present invention, wherein, a. Extraction solvent, b. Temperature of the extraction solvent, c. Extraction sampling time;

[0027] Figure 4 Quantitative detection of PPA in the breath of HPA patients and healthy volunteers provided by the present invention, wherein, a. LC-MS / MS is used to detect PPA; b. Comparison chart of PPA intensity and PPA at different concentrations;

[0028] Figure 5 Correlation analysis of the breath and blood of HPA patients provided by the present invention, wherein, a. Differential analysis of the breath PPA of HPA patients and healthy volunteers; b. Differential analysis of blood Phe of HPA patients and healthy volunteers; c. Correlation analysis of the breath PPA of HPA patients and blood Phe PPA. Detailed implementation manners

[0029] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0030] Refer to Figure 1 , the present invention provides a breath sampler, including a gas inlet channel, a gas collecting bottle, and a gas outlet channel. Among them, the gas inlet channel is used for the user to exhale into the sampler. The gas collecting bottle is equipped with a sealing cover and contains an extraction solvent for extracting exhaled gas inside. The inlet end of the gas inlet channel is provided with an interface adapted to the shape of the human mouth and nose to facilitate the user's exhalation. The outlet end of the gas inlet channel is located inside the gas collecting bottle and below the liquid level of the extraction solvent, and is used to extract the metabolic component PPA in the exhaled gas. The gas outlet channel is used to discharge the processed gas out of the sampler. The inlet end of the gas outlet channel is located inside the gas collecting bottle and above the liquid level of the extraction solvent. Check valves are respectively arranged on the gas inlet channel and the gas outlet channel to prevent gas backflow. A flow valve is arranged on the gas outlet channel to measure the flow rate of the exhaled gas, and by controlling the blowing speed of the exhaled gas, to ensure that the exhaled gas is in full contact with the extraction solvent and improve the extraction effect. The gas inlet channel, the gas collecting bottle, and the gas outlet channel are connected by a detachable connection method, which is convenient for the assembly and cleaning of the device.

[0031] The present invention provides a method for detecting exhaled gas based on a breath sampler

[0032] 1 Chemicals and materials

[0033] The phenylpyruvic acid standard (purity > 99.5%) was purchased from Tianjin Aladdin Chemistry Co., Ltd. (Tianjin, China). Absolute ethanol (HPLC grade) and methanol (HPLC grade) were purchased from Merck (USA Millipore); formic acid (HPLC grade) was purchased from Fisher. All experimental water was prepared using the milliq pure water system of Millipore Company (USAMillipore). A vortex oscillator (XH-B, Jiangsu Tianling) and an ultrasonic cleaner (JP-010T, Shenzhen Jiemeng) were also used for sample pretreatment in this work. The collection and preparation of exhaled breath and blood samples were carried out in the Key Laboratory of Birth Defect Prevention and Control in Jiangxi Province, Jiangxi Maternal and Child Health Hospital (Nanchang, China). All HPA patients were diagnosed by genetic testing according to our previous work (see Table 1). This study was approved by the Ethics Committee of the Key Laboratory of Birth Defect Prevention and Control in Jiangxi Province, Jiangxi Maternal and Child Health Hospital (Nanchang, China).

[0034] 2 Exhaled breath collection and preparation

[0035] To avoid interference and contamination of exhaled breath, oral gas was exhaled before exhaled breath sampling. After each subject took a deep breath for 5 s, exhaled breath was blown out at a flow rate of 0.25 L / min and quantitatively sampled through a centrifuge tube containing 10 ml of absolute ethanol solution as the adsorption solution ( Figure 1 ). The basic exhaled breath sample was obtained by repeating exhalation 6 times, with an interval of about 5 s between each exhalation, for a total of 1 min. Then the exhaled breath fluid was directly introduced into ESI-MS / MS and LC-ESI-MS / MS for mass spectrometry analysis.

[0036] 3 Blood collection and analysis

[0037] All blood samples were collected and prepared in the form of dried blood spots. Specifically: In the Key Laboratory of Birth Defect Prevention and Control in Jiangxi Province, Jiangxi Maternal and Child Health Hospital (Nanchang, China), 50 μL of whole blood from healthy people and HPA patients was pipetted onto filter paper (i.e., Guthrie card) to generate blood spot samples. The diameter of the spots was about 1.2 cm and they were air-dried at room temperature. After drying, the spots were manually punched and then transferred to 1.5 ml Eppendorf tubes. The spots were extracted with 500 μL of the stock solution containing all stable isotope-labeled standards in an ultrasonic bath for 30 min. The extract was ultrafiltered using an ultrafilter. The filtrate was diluted with 1 ml of methanol and then transferred onto a strong anion exchange phase for amino acid desalting.

[0038] 4 ESI-MS / MS and LC-ESI-MS analysis of exhaled breath extracts and blood extracts

[0039] To qualitatively identify respiratory PPA in the initial ESI-MS / MS experiment, high-resolution ESI-MS (Orbitrap QE, Thermo Fisher Scientific, Bremen, Germany) was used under conventional conditions: the ESI high voltage was 3.5 kV. The capillary temperature was set at 200 °C. Precursor ions were selected for MS / MS experiments, with an isolation window of 0.4 Da and a collision energy of 40%. Quantitative analysis of the exhaled extract was performed using a Waters ACQUITY UPLC BEH C18 (2.1 mm × 100 mm, 1.7 μm) liquid chromatography system coupled with a triple quadrupole mass spectrometer. Using the liquid chromatography system, mobile phase A: 0.1% formic acid in water, mobile phase B: methanol, flow rate 0.35 mL / min; injection volume: 10 μL; column temperature: 35 °C; gradient elution program: 0 - 1 min, 98% A; 1 - 2 min, 98% - 2% A; 2 - 4 min, 2% A; 4 - 4.3 min, 2% - 98% A; 4.3 - 6 min, 98% A. Using ESI-MS: ESI voltage: 4.5 kV; ESI temperature: 550 °C; nebulizing gas: 70 psi; auxiliary heating: 10 l / min; MS scan mode: negative ion scan; detection mode: multiple reaction monitoring (MRM) mode; curtain gas: 3.0 L / min; collision gas: 0.3 mL / min.

[0040] An ESI-MS / MS analysis of the blood extract was performed using a PE Sciex AB3200 (PE Sciex, Toronto, Canada) bench-top triple quadrupole mass spectrometer equipped with an ESI source. The probe operated at an ESI voltage of 5.0 kV. The ESI temperature was set at 100 °C. The curtain gas back pressure was 0.14 Mpa (20 psi), the nebulizer gas back pressure was 0.42 MPa (60 psi), and the turbo gas back pressure was 0.21 MPa (30 psi). A 20 μl sampling loop was installed on the injector; the methanol solvent flow rate was 50 μL / min. The peak intensity was determined by averaging the plateau of the total ion current. Data were acquired and processed using Mass Chrom 1.1 software (PE Sciex, Toronto, Canada), and data interpretation was performed using Multiview 1.4. MS / MS used multiple reaction monitoring (MRM) technology. PPA detection used a commercial non-derivatized tandem mass spectrometry screening kit (NeoBase TM Non-derivatized MS / MS kit (perkins-elmer Sciex)). In the MRM experiment, the source parameters, gas flow rates, and collision energy values of PPA were optimized using this kit.

[0041] 5 Results

[0042] 5.1 Development and Characterization of the Respiratory Sampler

[0043] To characterize the new breath sampler method, PPA standards were collected in a nitrogen stream (10 ng / mL) for 3 minutes. As a result, the PPA flowed into the selected extraction solvent and thus dissolved in the extraction solvent. The extraction solvent and PPA were analyzed using the high-resolution ESI-MS negative ion detection mode. As Figure 2 shown in a, PPA (C9H8O3, monoisotopic mass: 164.0474 Da) was recorded as a deprotonated ion ([m-h], m / z 163.0386) in the full mass spectrum and generated two major characteristic fragment ions at m / z 91.0540 - and m / z 135.0441. The peak at 135.0441 was attributed to the loss of CO in PPA, while the peak at m / z 91.0540 was a typical benzene anion, probably formed by further simple dissociation reactions due to the successive loss of CO (44 Da) in the fragment ion at m / z 135.0441. The benzyl anion not only provided characteristic ions for the identification of PPA but also provided an ion pair (m / z 163 > m / z 91) for the quantitative detection of PPA from complex breath matrices. Figure 2 b is the full mass spectrum of the detection of PPA in the breath extract of HPA patients, and there are abundant breath metabolites in the extract. Despite the complex breath matrix, PPA ( Figure 2 b) could be found through MS / MS experiments, which was in good agreement with the detection results of the standard PPA sample ( Figure 2 a). Therefore, these data clearly show that the breath PPA can be successfully collected using the breath sampler of the present invention and detected by conventional ESI-MS, which provides additional advantages for high-throughput analysis with a coupled LC system.

[0044] To further optimize the sampling performance of breath PPA, the sampling conditions were studied by detecting trace PPA in gas flow. The effects of extracting PPA with different solvents (pure water, methanol, ethanol / water (75 / 25, v / v), and pure ethanol) of the same volume were compared. It was found that pure ethanol had the best extraction efficiency ( Figure 3 a), probably because PPA has properties similar to ethanol. Considering the practical application in a real environment, temperature significantly affects gas-liquid extraction. Therefore, we studied the sampling temperature of the extraction solvent from room temperature to near boiling point, and the PPA signal increased with the increase in temperature ( Figure 3 b) because the hot solvent has greater solubility. We found that more PPA could be extracted into the extraction solvent as the sampling time increased from 0.5 min to 3.0 min ( Figure 3c). However, when the sampling time reaches 3 min, the PPA does not increase, mainly because the soluble PPA reaches an equilibrium state. Although PPA continues to be blown into the extraction solvent, PPA will also diffuse with the air flow, keeping the soluble PPA in the solution in an equilibrium state.

[0045] 5.2 Detection of Respiratory PPA by Sampler Coupled with LC-ESI-MS / MS

[0046] Under the optimized conditions, trace PPA was quantitatively analyzed by LC-ESI-MS / MS. Figure 4 a shows the LC chromatogram of PPA using the ion pair (m / z 135>m / z 91). The signal response diagram of PPA versus concentration shows a good linear relationship in the range of 0.05 - 1.0 ng / mL, with a linear correlation coefficient (R2 = 0.9968). Figure 4 b). The limit of detection (LOD) (S / N = 3) and limit of quantification (LOQ) of PPA were 0.01 ng / mL and 0.04 ng / mL, respectively. The relative standard deviations (rsd) of PPA in the extraction solvent were 6.18% (0.2 ng / mL) and 7.32% (1.0 ng / mL), respectively. The detection recoveries of PPA at different concentrations were 83.34% (0.2 ng / mL) and 81.73% (1.0 ng / mL), indicating that this method for detecting respiratory PPA is reliable and stable.

[0047] To explore the possibility of evaluating HPA by exhaled breath analysis, LC-ESI-MS / MS was used to detect the ion pair (m / z163bb0 m / z 91) for quantitative detection of exhaled PPA in HPA patients and healthy volunteers, as shown in Table 1. The data clearly show that trace exhaled PPA from HPA patients and volunteers can be sensitively quantified, so exhaled PPA is considered a potential indicator for evaluating the status of HPA. These analysis data indicate that the combination of a respiratory sampler and LC-ESI-MS / MS has the potential for enhanced detection of healthy PPA with high sensitivity, high specificity, and high speed.

[0048] Table 1

[0049]

[0050] 5.3 Correlation Analysis between Respiratory PPA and Blood Phe

[0051] The molecular mechanism of HPA is that phenylalanine (Phe) cannot be converted into tyrosine due to the lack of phenylalanine hydroxylase or tetrahydrobiopterin, and is abnormally metabolized to PPA in the human body. Therefore, in HPA patients, Phe accumulates in the blood, tissues, brain and other parts of the body. Therefore, detecting blood Phe is a method for clinical diagnosis of HPA. In addition, one of the abnormal metabolites of Phe, PPA, can also be detected in urine, skin and breath, presenting as a typical musty smell. To further understand the breath PPA in HPA patients, this study compared the detection results of breath PPA and blood Phe. As Figure 5 shown in a, there were significant differences in blood Phe between HPA patients and healthy volunteers, clearly indicating that Phe might accumulate in the blood (p < 0.001). Figure 5 b shows the comparison of breath PPA between HPA patients and healthy volunteers, and the difference was statistically significant (p < 0.01). Therefore, these data strongly suggest that the level of PPA in breath might be related to Phe in the blood. Figure 5 c shows the correlation analysis between exhaled PPA and blood Phe, and the correlation coefficient was 0.6262. Although the correlation coefficient was relatively low in this dataset (n = 19), this work still indicates a potential correlation between Phe accumulation and PPA metabolism. However, to better understand HPA, more research and a larger sample size are needed.

[0052] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A breath sampler, characterized in that: It includes a gas inlet channel, a gas collecting bottle and a gas outflow channel which are connected in sequence, wherein the gas inlet channel and the gas outflow channel are respectively provided with a one-way valve, the gas collecting bottle is equipped with a sealing cover and contains an extraction solvent, the outlet end of the gas inlet channel and the inlet end of the gas outflow channel are both located inside the gas collecting bottle, and the outlet end of the gas inlet channel is below the liquid surface of the extraction solvent, and the inlet end of the gas outflow channel is above the liquid surface of the extraction solvent.

2. The breath sampler according to claim 1, characterized in that The inlet end of the gas inflow channel is provided with an interface adapted to the shape of the human mouth and nose.

3. The breath sampler according to claim 1, characterized in that A flow valve is provided on the gas outflow channel.

4. The breath sampler according to claim 1, characterized in that The extraction solvent is ethanol.

5. The breath sampler according to claim 1, characterized in that: The gas inflow channel, the gas collecting bottle and the gas outflow channel are connected in a detachable connection manner.

6. A method for detecting exhaled gas based on the breath sampler according to any one of claims 1 to 5, characterized in that: The method comprises: Blow out the exhaled gas from the inlet end of the gas inflow channel and collect the exhaled liquid in the gas collecting bottle; The exhaled liquid is detected by LC-MS / MS instrument to obtain the exhaled gas PPA concentration.

7. The exhaled gas detection method according to claim 6, characterized in that: After taking a deep breath for 5 seconds, blow out the exhaled air at a flow rate of 0.2-0.3L / min, and blow out the exhaled air 6 times.

8. The exhaled gas detection method according to claim 6, characterized in that: The exhalation time is 1 minute.

9. The exhaled gas detection method according to claim 6, characterized in that: The ESI voltage was 4.5 kV, the ESI temperature was 550°C, the nebulizer gas was 70 psi, the auxiliary heating was 10 L / min, the curtain gas was 3.0 mL / min, and the collision gas was 0.3 mL / min.

Images