CC BY
21DOI: 10.6060/ivkkt.20216402.6294
ОДНОВРЕМЕННЫЙ КОЛИЧЕСТВЕННЫЙ XH ЯМР АНАЛИЗ МЕТАНОЛА, ЭТАНОЛА И ИХ МЕТАБОЛИЧЕСКИХ ПРОДУКТОВ В ПЛАЗМЕ ЧЕЛОВЕКА: РАННИЙ ДИАНОЗ И МОНИТОРИНГ ВО ВРЕМЯ ЛЕЧЕНИЯ ОСТРОГО ОТРАВЛЕНИЯ МЕТАНОЛОМ
ВО ВЬЕТНАМЕ
Та Тхи Тхао, Нгуен Тхи Нган, Ву Ан Фуонг, Ха Тран Хунг, Нгуен Ван Тхык, Фам Куанг Трунг
Та Тхи Тхао, Нгуен Ван Тхык, Фам Куанг Трунг*
Химический факультет, Университет естественных наук, Ханойский государственный университет, Ле Тхан Тонг, 19, Хоан Кием, Ханой, Вьетнам, 110401
E-mail: [email protected], [email protected], [email protected]* Нгуен Тхи Нган, Ву Ан Фуонг
Центр контроля ядов, Больница Бах Май, Ханой, Вьетнам, 116301 E-mail: [email protected], [email protected]
Ха Тран Хунг
Отделение неотложной помощи, интенсивной терапии и токсикологии, Ханойский медицинский университет, тон Тхат Тунг, 1, Донг да, Вьетнам, 116001 E-mail: [email protected]
В данной работе был установлен метод ядерного магнитного резонанса (Н-ЯМР) для одновременного количественного определения метанола, этанола и продуктов их метаболизма (муравьиная кислота и уксусная кислота) в плазме человека. Эффект белка в образце плазмы был удален 25% трихлоруксусной кислотой (TCA) с соотношением TCA и образца плазмы при 1/5 (об./об.). Небольшое количество D2O (приблизительно 1/10 об./об.) было выбрано из-за того, что влияние водяного сигнала было устранено с помощью предварительного подавления воды с помощью последовательности импульсов NOESY. Были проверены различные аналитические параметры, такие как линейность, точность, точность и специфичность, предел обнаружения прибора (IDL) и предел количественного определения прибора (IQL). Предел обнаружения прибора четырьмя соединениями в пустой плазме варьировался от 0,68 (для метанола) до 2,88 мг/л (для муравьиной кислоты). Среднее извлечение концентрации в динамических диапазонах составило от 96 до 105%. Хорошая линейность между концентрацией, рассчитанной по интегральному сигналу, и измеренной, полученной по калибровочным кривым, подтвердила, что концентрацию анализируемых веществ в плазме можно непосредственно определить из сигнала протона, полученого из спектроскопии ядерного магнитного резонанса (1Н-ЯМР). В данной работе предложенный метод был успешно применен для прямого определения пяти соединений в 9 образцах плазмы пациента с удовлетворительными результатами по сравнению с методом GC/FID. Аналитическая процедура может быть полезна для диагностики и оценки лечения отравления метанолом во вьетнамских больницах.
Ключевые слова: метанол, этанол, Вьетнам, магнитный, протон Для цитирования:
Та Тхи Тхао, Нгуен Тхи Нган, Ву Ан Фуонг, Ха Тран Хунг, Нгуен Ван Тхык, Фам Куанг Трунг Одновременный количественный 1H ЯМР анализ метанола, этанола и их метаболических продуктов в плазме человека: ранний диа-ноз и мониторинг во время лечения острого отравления метанолом во Вьетнаме. Изв. вузов. Химия и хим. технология. 2021. Т. 64. Вып. 2. С. 41-48 For citation:
Ta Thi Thao, Nguyen Thi Ngan, Vu Anh Phuong, Ha Tran Hung, Nguyen Van Thuc, Pham Quang Trung Simultaneous quantitative 1H NMR analysis of methanol, ethanol and their metabolic products in human plasma: early dianosis and monitoring during treatment of acute methanol poisoning in Vietnam. Izv. Vyssh. Uchebn. Zaved. Khim. Khim. Tekhnol. [ChemChemTech]. 2021. V. 64. N 2. P. 41-48
SIMULTANEOUS QUANTITATIVE 1H NMR ANALYSIS OF METHANOL, ETHANOL AND THEIR METABOLIC PRODUCTS IN HUMAN PLASMA: EARLY DIANOSIS
AND MONITORING DURING TREATMENT OF ACUTE METHANOL POISONING IN VIETNAM
Ta Thi Thao, Nguyen Thi Ngan, Vu Anh Phuong, Ha Tran Hung, Nguyen Van Thuc, Pham Quang Trung
Ta Thi Thao, Nguyen Van Thuc, Pham Quang Trung*
Faculty of Chemistry, VNU University of Science - Hanoi, Le Thanh Tong Str., 19, Hanoi, 110401, Vietnam
E-mail: [email protected], [email protected], [email protected]*
Nguyen Thi Ngan, Vu Anh Phuong
Poison Control Center, Bach Mai Hospital, Hanoi, 116301, Vietnam
E-mail: [email protected], [email protected]
Ha Tran Hung
Department of Emergency, Intensive Care Medicine and Toxicology, Hanoi Medical University, Ton That Tung,
1, Dong Da, Hanoi, 116001, Vietnam
E-mail: [email protected]
The 1H-NMR methodfor simultaneous quantification of methanol, ethanol and their metabolic products (formic acid and acetic acid) in human plasma was established. The effect of protein in plasma sample was removed by 25% trichloroacetic acid (TCA) with the ratio of TCA and plasma sample at 1/5 (v/v). The small amount of D2O (estimated 1/10 v/v) was chosen due to the effect of water signal was eliminated by using the advance water suppression with NOESYpulse sequence. The different analytical parameters such as linearity, precision, accuracy, and specificity, instrument detection limit (IDL) and instrument quantification limit (IQL) were validated. The instrument detection limit offour compounds in blank plasma varied from 0.68 (for methanol) to 2.88 mg/L (for formic acids). The average recoveries of concentration in dynamic ranges were found to be 96 to105%. The good linearity between concentration calculated by integrated signal and measured obtained by calibration curves confirmed that concentration of analytes in plasma can be directly determined from the 1H-NMR signal. The proposed method has been successfully applied for direct determination offive compounds in 9 patient's plasma samples with the satisfactory results compared to GC/FID method. The analytical procedure can be usefulfor diagnosis and evaluation of treatment of methanol poisoning in Vietnamese hospitals.
Key words: methanol, ethanol, Vietnam, magnetic, proton
INTRODUCTION
In recent years, the methanol poisoning in Vietnam is increasing with a large number of poisonous patients. In 2014, there were only 16 methanol poisoning patients transferred to Poison Control Center -Bach Mai hospital, but it reached 33 and 46 patients in 2015, 2016, and it was 48 patients within the first six months of 2017. It is alarming that the group poisoning cases (40 methanol poisoning victims and 9 deaths) happened in Lai Chau or Gia Lai province in 2017. Practically, the early diagnosis [1, 2] of methanol poisoning is difficult because that clinical symptom of methanol poisoning is quite similar to other ones such as those of ethanol, ethylene glycol, and isopropanol poisoning. This led to the delay application of the specific treatments for the patients that resulted in a relatively high mortality rate of 18-54%. The toxicity of
methanol for human is caused by very toxic formic acid formed from formaldehyde which is metabolite of methanol [3, 4]. In methanol poisoning treatment, eth-anol is used as antidote for limiting the metabolism to formic acid, and then methanol and ethanol should be eliminated by haemodialysis. Therefore, an early monitoring of ethanol, methanol and their metabolic products simultaneously in human plasma just after patients enter to the hospital and during treatment is very important in methanol poisoning treatment.
Traditionally, the simultaneous quantification of methanol, ethanol and selected metabolic products can be done by UV-VIS[5], Raman spectroscopy [6, 7], GC-MS [8, 9], GC-FID [10-12], eletropheresis[13] or cyclic voltammetry [14]. These methods have good sensitivity, but they often require highly skilled analysts and complicated sample preparation [15].
Nuclear magnetic resonance (NMR) spectroscopy is gaining more and more application in quick, simple and non-destructive analysis of samples with complicated matrix, whereas it can allow simultaneously determine different compounds in a mixture without sample preparation and separation before quantification [16-18]. Accepting the NMR detection limits, blood is still a complex mixture that, in addition to this complication, presents additional challenges. The presence of protein and other components in blood and plasma can affect to the quantification of selected compounds [19, 20] when several signals overlap, particularly those of ethanol and glucose. This phenomenon could result in disadvantages of 1H-NMR quantitative analysis in biological samples [19, 21, 22]. A simple deproteinization and solvent suppression step can not only improve the spectrum resolution, but also simplify the quantification analysis.
In this research, by setting up a suitable procedure for deproteinization and water suppression by advance NOESY pulse sequence, a simple, rapid and inexpensive procedure utilizing quantitative 1H-NMR (qHNMR) with solvent suppression was established to quantify simultaneously methanol, ethanol and their aldehyde- and acid-metabolic products in human plasma. The obtained results by the proposed method can be applied for rapid diagnosis of methanol poisoning as well as its treatment monitoring.
MATERIALS AND METHODES
Reagents, Reference and Standard solutions
LC grade chemicals namely methanol (99.99%), ethanol (99.99%), formaldehyde (solution, 38%), acetaldehyde, formic (solution, 90%) and acetic acid (100%) obtained from Merck (US) were used for preparing stock standards in deionized water. Working calibration standard solutions were daily prepared by dilution of stock standards in deionized water. Maleic acid (MA) (99.94% deuterated) purchased from Sigma-Aldrich (Singapore) and deionized water was used for preparing the stock internal standard. Deuterium oxide D2O (Armar Chemical) was used as solvent for qHNMR. Tetramethylsilane (TMS, 99% deuter-ated) purchased from Sigma-Aldrich (Singapore) was used as NMR reference to correct resonance chemical shifts (S in ppm). A 25% (v/v) trichloroacetic acid (TCA) solution was used for deproteinization of the plasma samples.
Sampling and sample preparation
To ensure the efficiency and accuracy of analytical method, plasma samples obtained from healthy people in Vietnam Institute of Haematology and Blood
Transfusion (Hanoi, Vietnam) were use as blank samples and standard reference (SRM) samples (by spiking known amount of standards into blank samples).
Nine blood samples of patients infected by methanol poisoning were collected from methanol poisoning patients (in 2018) in Poison Control Center of Bach Mai Hospital, Hanoi and transferred into plastic tubes containing heparin. The samples were then cen-trifuged at 4000 rpm for 15 minutes. The upper plasma was taken into capped vial and stored at -4 °C. 0.2 mL of 25% trichloroacetic acid (TCA) solution was added to 1 mL of protein-separated plasma, then the sample was again centrifuged for 10 minutes at 4000 rpm, and the supernatant was taken for NMR analysis. The methanol concentrations in blood samples determined by GC/FID at Department of Chemical Forensic Medicine - National Institute of Forensic Medicine (Hanoi, Vietnam) were used as references data.
qHNMR measurement
Analytical samples were analyzed in the standard 5 mm NMR tubes. 0.9 mL of sample were mixed with 0.1 mL of D2O containing tetramethylsilane (TMS). The 1H-NMR spectra were acquired at 300 K on a Bruker Avance III 500 MHz (at Faculty of Chemistry, VNU University of Science, Vietnam National University, Hanoi) equipped with a 5 mm BBFO probe including automated locking, matching, tuning and calibration of the 90° hard pulse and operating at 11.7 T. The NMR protocol applied was based on the Bruker standard protocol NOESYGGPPR1D established for solvent suppression using advance NOESY sequence. The two successive 1H-NMR experiments used for the acquisition of each sample were as follows:
Experiment 1 (measure the exact resonance frequency of water using standard water suppression, Bruker sequence ZGPR): First, a one-scan standard 1H NMR spectrum was recorded. The peak of water at around 4.7 ppm was chosen as offset frequency o1. Then, the exact frequency of water in each sample was determined by using a standard Bruker water presatu-ration pulse program ZGPR. This pulse program only suppresses the signal of OH protons by applying a pre-saturation scheme with low-power o1 frequency irradiation. In the experiment, a 25-Hz RF field was used for the presaturation, the relaxation delay (RD) and acquisition time (AQ) were set to 4 s and 3.27 s, respectively, resulting in a total recycle time of 7.27 s. Two dummy scans (DS) were applied, and four free induction decays (FIDs) (number of scans, NS = 4) were collected into a time domain (TD) of 65536 (65 k) complex data points using a 19.9947 ppm spectral width and a receiver gain (RG) of 3.2. The FID signals were
multiplied with an exponential function, which corresponded with line broadening 0.3 Hz prior to Fourier transformation. Repeat this step until the OH signal of water was totally suppressed to a negative peak (Fig. 1). This frequency was used for optimization the shape pulse that is required for the advance water suppression in second experiment.
Experiment 2 (advance water suppression using NOESY pulse sequence - NOESYGPPR1D). The parameters RD, P(90°), AQ and TD were set the same asin Experiment 1; DS was 4, NS was 32 and the mixing time was set to 100ms. The optimum RG was set to 20.2, which resulted in a considerable gain in signal-to-noise ratio (S/N) compared with Experiment 1 (with pulse sequence ZGPR). The shape pulse was calculated using command pulsecal in Topspin 3.2. Total time required for analyses was about 10 minutes per sample.
All 1H-NMR were automatically baseline corrected using the Topspin 3.2 package (Bruker Biospin, Germany) by the command absn.
For quantification, NMR signals in the specific regions for each compound were integrated and summarized. Samples were normally quantified using linear calibration curves. Besides, the concentration of X compound could be also directly calculated from equation 1, and were compared with the concentrations determined by standard calibration curves.
* AjS Nx Mis Vx lS W
where Cx and CIS were concentration of X and internal standard MA, A was the peak integration, M was the molar mass, N was the number of resonant protons, and V was the volume of X and internal standard.
Validation of analytical method
The linearity was studied in a concentration range of 15.00 mg/L to 10.50 mg/mL of each compound in a mixture of methanol, ethanol, acetaldehyde, formic acid and acetic acid in water. The mixture of 0.6 mL of solution of 5 substances at different concentrations, 0.3 mL of 200 mg/L MA solution and 0.1 mL D2O was analysed by 1H-NMR. The method validation for four analytes (methanol, ethanol, formic acid and acetic acid) was conducted using blank and spiked plasma samples.
Signal-to-noise calculation
To quantitatively evaluate the enhancement in intensity (the amplitude of the highest peak in the signal region) and signal-to-noise ratio (SNR), the Bruker sino routine was applied to determine SNR [23]. The sino command in the TopSpin 3.2 software package gives the SNR of a 1D spectrum evaluated by the formula SNR = maxval/(2*noise), where maxval is the highest intensity in the spectral region and noise is the
average level of noise in the noise region. The instrument detection method (IDL) and instrument quantification limit (IQL) were calculated by equations:
I DL = 3 X SNR / b (2)
IQL = 10 X SNR / b (3)
where b is the slop of calibration curve. The line broadening of all spectra was set at 0.3 Hz, and all spectra were automatically shimming by command topshim tunea to ensure the same shim quality.
RESULTS AND DISCUSSION
Optimization of sample preparation and HNMR measurement conditions
The optimization of the sample preparation and measurement protocol was the key factor for the development of the quantitative NMR analyses, which should be applicable for the complicated blood matrix. In order to simplify the sample preparation, water itself in blood acted as solvent and only 10% of D2O were added. Due to the very strong signal of water that hindered all the small signals (Fig. 1-(1)), a solvent suppression protocol was needed. The simplest solvent suppression (ZGPR in Bruker's standard library) uses a long continuous but weak intensity pulse that have same frequency with resonance frequency of water just before the main pulse (water presaturation). The method could suppress not only part of water signal, but also the proton exchange signal of OH, NH groups [23, 24]. Therefore, the signal of formic acid was partially eliminated (Fig. 1-(2)). Another NOESYGPPR1D solvent suppression method applied the water presatu-ration scheme in combination with NOESY pulse (NOE: Nuclear Overhausser Effect) was conducted for observing NH and OH group signals. Pulse program allows observing all signals that have strong proton exchange in the water medium, hereby the CH group of formic acid (Figure 1-(3)). Practically, it is possible to directly quantify five compounds around the suppressed water signal. Each sample underwent an automatic tuning of the probe head, followed by an optimization of the power for water saturation and pulse determination using pulsecal command in Topspin. As none signal selected for quantification of the target compounds was very close to water suppressed signal, no correction was necessary. The performance of solvent suppression NOESYGPPR1D allowed the used of water as solvent, so only 10% of D2O needed for frequency calibration, that reduced the cost of experiment.
By using NOESYGPPR1D pulse, the measurement of the standard solutions was relatively simple with no peak overlapping observed. The chemical shifts (Table 1) of methanol, ethanol and their respective aldehyde and acid metabolites except formaldehyde showed a clearly separation and could be used for
their simultaneous quantification in a mixture. Under the experimental condition, the 'H - NMR spectra of a plasma sample without protein precipitation also clearly showed the methanol and formic acid signals, but the CH3 signals of ethanol and acetic acid were overlapped (Fig. 2-(3)), and quantification of formaldehyde in a mixture was impossible. But due to very short life-time of formaldehyde and acetaldehyde in blood [2] (change to formic acid and acetic acid after that) so the determination of aldehydes in this study was ignored.
Table 1
Chemical shift of five compounds in standard solution
(in water) and in plasma samples [22, 25] Таблица 1. Химический сдвиг пяти соединений в
Chemical shift in Chemical shift in
Compound Signal standard solutions (ppm) plasma samples (ppm)
Methanol CH3 (s) 3.37 3.20
Ethanol CH3 (t) 1.19* 1.06*
CH2 (q) 3.54 3.47
Acetalde- CH3 1.22
hyde (m)
Acetic acid CH3 (s) 2.09 1.93
Formaldehyde CH (s) 9.60 -
Formic acid CH (s) 8.27 8.08
Maleic acid CH (s) 6.35 6.31
Notes: s): singlet, (t): triplet, (q): quadruplet, (m): multiplet (*: The chemical shift was used for quantification of ethanol) Примечания: синглет, (t): триплет, (q): квадруплет, (m): мультиплет
(*: Химический сдвиг использовался для количественного определения этанола)
5.0 4.0 3.0 fl (ppm)
Fig. 1. The 'H- NMR spectra of a standard solution containing 5 ana-lytes (1) without solvent suppression, (2) solvent suppression by ZGPR pulse program and (3) solvent suppression by NOE-SYGPPR1D pulse program Рис.1. Спектры 'H-ЯМР стандартного раствора, содержащего 5 аналитов: (1) без подавления растворителя, (2) подавление растворителя с помощью импульсной программы ZGPR и (3) подавление растворителя с помощью импульсной программы NOESYGPPR1D
The spectra of plasma samples (with depro-teinization) showed good baseline and no peak overlapping of all studied signals (Fig. 2-(1)). The CH3 triplet signal of ethanol at 1.06 ppm was chosen for ethanol quantification study (Fig. 3). This procedure was used for subsequent experiments.
5.0 4.0
fl (ppm)
Fig. 2. •H-NMR spectra of spiked plasma sample (4) without solvent suppression but no deproteination, (3) solvent suppression by ZGPR pulse programbut no deproteination, (2) solvent suppression by NOESYGPPR1D pulse program but no deproteination, (1) solvent suppression by NOESYGPPR1D pulse program, with
protein precipitation Рис. 2. Спектры *Н-ЯМР пробы плазмы с добавкой: (4) без подавления растворителем, но без депротеинации, (3) подавление растворителя с помощью импульсной программы ZGPR, но без депротеинизации, (2) подавление растворителем с помощью импульсной программы NOESYGPPR1D, но без депротеинации, (1) подавление растворителем импульсной программой NOESYGPPR1D с осаждением белка
CH> OH
ChPCHO
HCOOH C№ C№OI
1 _J|jLL hi 11 1
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 Э.О 2.5 2.0 1.5 1.0 0.5 0.1
Fig. 3. 'H NMR spectra of a spiked plasma sample (after protein precipitation) using NOESYGPPR1D pulse program Рис. 3. Спектры 'H-ЯМР пробы плазмы с добавкой (после осаждения белка) с использованием импульсной программы NOESYGPPR1D
Validation of analytical method
Linearity
The calibration curves were done based on signals of five standard solutions and spiked plasma sample containing of five or four compounds. The relationship between integrated peak areas and concentration showed good linearity with high correlation coefficient (R2value > 0.999) (Fig. 4a). The instrument detection method (IDL) and instrument quantification limit (IQL) of five compounds were calculated according to equations 2 and 3 base on signal-to-noise [23, 25]. Results were given in Table 2. Methanol determination exhibited the most sensitivity with the lowest IDL and
IQL of 0.02 mg/L and 0.07 mg/L respectively. The instrument detection limit (IDL) and instrument quantification limit (IQL) of four compounds in blank plasma was from 0.68 (for methanol) to 2.88 mg/L (for formic acid) and 2.25 (for methanol) to 9.60 mg/L(for formic acid), respectively.
The concentration of selected compounds in spiked samples evaluated by standard calibration curve is in good linear correlation with that by direct calculation using equation (1) (Table 2). It is clearly that methanol, ethanol, acetic acid and formic acid could be directly determined in plasma sample by using the integrated signal with the calibration factors.
25QQ
2QQQ
/S
1QQQ
5QQ
3QQ
4
5QQQ 1QQQQ
C, mg/L
15QQQ
5QQQ 1QQQQ C, mg/L b
15QQQ
Fig. 4. a: Calibration curves of 1-methanol, 2-ethanol, 3-formic acid, 4-acetaldehyde and 5-acetic acid in standard solutions. b: Calibration curves of 1 -methanol, 2-ethanol, 3-formic acid and 4-acetic acid in spiked plasma samples Рис.4. а: Градуировочные кривые для 1-метанола, 2-этанола, 3-муравьиной кислоты, 4-ацетальдегида и 5-уксусной кислоты в стандартных растворах. b: Калибровочные кривые 1-метанола, 2-этанола, 3-муравьиной кислоты и 4-уксусной кислоты в пробах плазмы с добавками
Table 2
IDL and IQL of selected compounds in standard solutions (water) and in plasma samples Таблица 2. IDL и IQL выбранных соединений в стандартных растворах (вода) и в образцах плазмы
Standard solutions Plasma samples
Analytes Correlation Coefficient be- IDL (mg/L) IQL (mg/L) Correlation Coefficient be- IQL (mg/L)
tween measured* and cal- tween measured* and cal- IDL (mg/L)
culated "concentrations culated** concentrations
Methanol 1.01 Q.Q2 Q.Q74 0.69 0.68 2.25
Ethanol 1.02 Q.Q3 Q.Q9 0.81 0.82 2.74
Acetaldehyde 0.95 0.10 Q.23 - - -
Formic acid 1.22 0.10 Q.324 0.67 2.88 9.6Q
Acetic acid 0.94 Q.Q3 0.11 0.96 0.91 3.Q4
1
5
2
3
Q
Q
Q
a
Note: (*concentration of analytes determined based on calibration curves; ** concentration of analytes directly calculated by using equation (1); «-» did not measure)
Примечания: (* концентрация аналитов, определенная на основании калибровочных кривых; ** концентрация аналитов, рассчитанная непосредственно с использованием уравнения (1); «-» не измерено)
Accuracy (precision and trueness) of the analytical method
The relative standard deviations (%RSD) were evaluated based on analyzing of replicate spiked samples and were below than 5% for all concentrations in the dynamic range. The percentage recoveries varied
in the range of 96-107% (Table 3) confirmed satisfactory reliability of the analysis.
Determination of methanol, ethanol and their metabolic products in plasma samples of patients
Nine plasma samples collected from 9 methanol poisoning patients (in 2018) in Poison Control
Center of Bach Mai hospital were analysed using the above proposed analytical procedure. It was confirmed that the patients were overexposure with methanol. They accidentally or deliberately ingested methanol as a substitute for ethanol.
The amount of methanol, ethanol, formic and acetic acid found in the samples by qHNMR compared with methanol and ethanol concentrations determined by GC/FID were given in the Table 4. Despite of the very complicated matrix of plasma, the successful quantification of all four substances in human plasma samples with acceptable deviation of 2-23%makes qHNMR possible method for a quick and precise determination of methanol in blood and treatment monitoring. The different ratios of formic acid to methanol concentration or of acetic acid to ethanol concentrations in patient's plasma samples suggested that the changes of methanol and ethanol to acids dependent on the duration since patients drunk alcohol. Therefore, the determination of formic acids and acetic acid to
Table 4
Concentrations of methanol, ethanol and their respective acids in 9 plasma samples Таблица 4. Концентрации метанола, этанола и их соответствующих кислот в 9 образцах плазмы
N Patient Methanol (mg/L) Ethanol (mg/L) Formic acid, mg/L Acetic acid, mg/L
NMR GC/FID Bias (%) NMR GC/FID Bias (%) NMR NMR
1 Case 1 9б9 S67 +12 427 514 -17 354 154
2 Case 2 2301 234S -2 N.D. N.D. - 779 -
3 Case 3 1045 1312 -20 539 571 -5 N.D. -
4 Case 4 2274 1973 +15 N.D N.D. - 1001 -
5 Case 5 N.D N.D - 4525 36S0 +23 N.D. 102
б Case б N.D. N.D. - 1954 1900 +3 N.D. 116
7 Case 7 473 407 +16 б00 5S0 +3 518 63.7
S Case S 2250 20б1 +9 7S7 794 -1 641 62.8
9 Case 9 40S 3бб +12 423 450 -6 104 182
gether with methanol and ethanol is necessary to helpful doctors to decide suitable treatments.
Table 3
Extraction and %RSD of four analytes in plasma samples Таблица 3. Извлечение и % RSD четырех аналитов __в образцах плазмы _
Concentration (mg/L) in spiked plasma samples RSD % Recovery percentage (%)
Methanol 79 3.95 101±2
790 4.б7 106±2
3950 1.90 100±1
Ethanol 79 4.79 99±2
790 4.75 105±2
3950 1.S3 100±1
Formic acid 108 3.55 97±2
1080 4.б2 104±2
5400 3.44 102±2
Acetic acid 105 2.б0 103±1
1025 4.13 108±2
5250 0.92 99±2
Note: (N.D. not detected; "-" could not be determined or calculated)
Примечания: (N.D. не обнаружен; "-" не могло быть определено или рассчитано)
CONCLUSION
A rapid qHNMR method for simultaneous quantification of methanol, ethanol and their acid metabolic products in plasma samples was developed, validated and applied for analysis of 9 patient's plasma samples. Compared to a traditional GC/FID method that needs derivation reactions of acetic acid and formic acid before chromatographic analysis, the quantitative 1H-NMR method with good linearity and accuracy was found to be more suitable for simultaneous determination of selected compounds. The simple plasma preparation using TCA deproteinization and
absolute quantification could make the proposed qHNMR method turn to be more comprehensive method rather than GC/FID that must use standard solutions for making calibration curves. Thus, qHNMR was shown as a good alternative method in the diagnosis and evaluation of methanol treatment poisoning in Vietnamese hospitals.
ACKNOWLEDGMENTS This research received the support from RoHan Catalysis project.
Это исследование было поддержано проектом RoHan Catalysis.
REFERENCES ЛИТЕРАТУРА
1. Clary J.J. Human Toxicity. In: The Toxicology of Methanol. Wiley. 2013. P. 269. DOI: 10.1002/9781118353110.
2. Hovda K.E., Mcmartin K., Jacobsen D. Methanol and Formaldehyde. In: Critical Care Toxicology: Diagnosis and Management of the Critically Poisoned Patient. Ed. by J. Brent. Cham: Springer International Publishing. 2017. P. 3058. DOI: 10.1007/978-3-319-20790-2_78-2.
3. BNF 69. British National Formulary. V. 42. British Medical Association. 2015.
4. Hovda K.E., Hunderi O.H., Tafjord A.-B., Dunlop O., Rudberg N., Jacobsen D. Methanol outbreak in Norway 2002-2004: epidemiology, clinical features and prognostic signs. J. Internal Medicine. 2005. V. 258. N 2. P. 181-190. DOI: 10.1111/j.1365-2796.2005.01521.x.
5. Jendral J.A., Monakhova Y.B., Lachenmeier D.W. Formaldehyde in Alcoholic Beverages: Large Chemical Survey Using Pur-pald Screening Followed by Chromotropic Acid Spectrophotome-try with Multivariate Curve Resolution. Internat. J. Analyt. Chem. 2011. V. 2011. P. 1-11. DOI: 10.1155/2011/797604.
6. Boyaci I.H., Genis H.E., Guven B., Tamer U., Alper N. A novel method for quantification of ethanol and methanol in distilled alcoholic beverages using Raman spectroscopy. J. Raman Spectrosc. 2012. V. 43. N 8. P. 1171-1176. DOI: 10.1002/jrs. 3159.
7. Ellis D.I., Muhamadali H., Xu Y., Eccles R., Goodall I., Goodacre R. Rapid through-container detection of fake spirits and methanol quantification with handheld Raman spec-troscopy. Analyst. 2019. V. 144. N 1. P. 324-330. DOI: 10.103 9/C8AN01702F.
8. Kage S., Kudo K., Ikeda H., Ikeda N. Simultaneous determination of formate and acetate in whole blood and urine from humans using gas chromatography-mass spectrometry. J. Chromatography B. 2004. V. 805. N 1. P. 113-117. DOI: 10.1016/j.jchromb.2004.02.029.
9. Arslan M.M., Zeren C., Aydin Z., Akcan R., Dokuyucu R., Ke-ten A., Cekin N. Analysis of methanol and its derivatives in illegally produced alcoholic beverages. J. Forensic Legal Medicine. 2015. V. 33. P. 56-60. DOI: 10.1016/j.jflm.2015.04.005.
10. De Paula Pereira P.A., Sousa Santos E.T., De Freitas Ferreira T. Determination of methanol and ethanol by gas chro-matrography following air sampling onto florisil cartridges and their concentrations at urban sites in the three largest cities in Brazil. Talanta. 1999. V. 49. N 2. P. 245-252. DOI: 10.1016/S0039-9140(98)00376-2.
11. Wang M.-L., Wang J.-T., Choong Y.-M. A rapid and accurate method for determination of methanol in alcoholic beverage by direct injection capillary gas chromatography. J. Food Composit. Anal. 2004. V. 17. N 2. P. 187-196. DOI: 10.1016/j.jfca.2003.08.006.
12. Zhuang G.-C., Lin Y.-S., Elvert M., Heuer V.B., Hinrichs K.-U. Gas chromatographic analysis of methanol and ethanol in marine sediment pore waters: Validation and implementation of three pretreatment techniques. Marine Chem. 2014. V. 160. P. 82-90. DOI: 10.1016/j.marchem.2014.01.011.
13. Santos M.S.F., Da Costa E.T., Gutz I.G.R., Garcia C.D. Analysis of Methanol in the Presence of Ethanol, Using a Hybrid Capillary Electrophoresis Device with Electrochemical
Derivatization and Conductivity Detection. Analyt. Chem. 2017. V. 89. N 2. P. 1362-1368. DOI: 10.1021/acs.anal-chem.6b04440.
14. Pereira P.F., Sousa R.M.F., Munoz R.A.A., Richter E.M. Simultaneous determination of ethanol and methanol in fUel ethanol using cyclic voltammetry. Fuel. 2013. V. 103. P. 725-729. DOI: 10.1016/j.fuel.2012.07.034.
15. Kew W., Bell N.G.A., Goodall I., Uhrín D. Advanced Solvent Signal Suppression for the Acquisition of 'D and 2D NMR Spectra of Scotch Whisky. Magn. Resonance Chem. 2017. V. 55. N 9. P. 784-784. DOI: 10.1002/mrc.4628.
16. Simmler C., Napolitano J.G., Mcalpine J.B., Shao-Nong Chen, Pauli G.F. Universal quantitative NMR analysis of complex natural samples. Curr. Opinion Biotechnol. 2014. V. 25. P. 51-59. DOI: 10.1016/j.copbio.2013.08.004.
17. Marcone M.F., Wang S., Albabish W., Nie Sh., Somnarain D., Hill A. Diverse food-based applications of nuclear magnetic resonance (NMR) technology. Food Res. Internat. 2013. V. 51. N 2. P. 729-747. DOI: 10.1016/j.foodres.2012.12.046.
18. Wu Z., Jin L., Zheng W., Zhang Ch., Zhang L., Chen Y., Guan J., Fei H. NMR-based serum metabolomics study reveals a innovative diagnostic model for missed abortion. Bio-chem. Biophys. Res. Commun. 2018. V. 496. N 2. P. 679-685. DOI: 10.1016/j.bbrc.2018.01.096.
19. Fan T.W.M., Lane A.N. Applications of NMR spectroscopy to systems biochemistry. Prog. Nucl. Magn. Resonance Spectrosc. 2016. V. 92-93. P. 18-53. DOI: 10.1016/j.pnmrs.2016.01.005.
20. Tukiainen T., Tynkkynen T., Mäkinen V. -P. A multi-metabolite analysis of serum by NMR spectroscopy: Early systemic signs of Alzheimer's disease. Biochem. Biophys. Res. Commun. 2008. V. 375. N 3. P. 356-361. DOI: 10.1016/j.bbrc.2008.08.007.
21. Nagana Gowda G.A., Gowda Y.N., Raftery D. Expanding the Limits of Human Blood Metabolite Quantitation Using NMR Spectroscopy. Analyt. Chem. 2015. V. 87. N 1. P. 706-715. DOI: 10.1021/ac503651e.
22. Wahl A., Azaroual N., Imbenotte M., Mathieu D., Forzy G., Cartigny B., Vermeersch G., Lhermitte M. Poisoning with methanol and ethylene glycol: *H NMR spectroscopy as an effective clinical tool for diagnosis and quantification. Toxicology. 1998. V. 128. N 1. P. 73-81. DOI: 10.1016/S0300-483X(98)00055-9.
23. Monakhova Y.B., Schäfer H., Humpfer E., Spraul M., Ku-balla T., Lachenmeier D.W. Application of automated eightfold suppression of water and ethanol signals in 1H NMR to provide sensitivity for analyzing alcoholic beverages. Magn. Resonance Chem. 2011. V. 49. N.11. P. 734-739. DOI: 10.1002/mrc.2823.
24. Sedaghat Doost A., Akbari M., Stevens C.V., Setiowati A.D., Van der Meeren P. A review on nuclear overhauser enhancement (NOE) and rotating-frame overhauser effect (ROE) NMR techniques in food science: Basic principles and applications. Trends FoodSci. Technol. 2019. V. 86. P. 16-24. DOI: 10.1016/j.tifs.2019.02.001.
25. Zailer E., Diehl B.W.K. Alternative determination of blood alcohol concentration by 1H NMR spectroscopy. J. Pharm. Biomed. Anal. 2016. V. 119. P. 59-64. DOI: 10.1016/j.jpba.2015.11.030.
Поступила в редакцию 23.07.2020 Принята к опубликованию 03.12.2020
Received 23.07.2020 Accepted 03.12.2020
