药物检测中同步荧光光谱法的应用研究
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摘要
荧光分析法以其灵敏度高、选择性好、取样量少、仪器设备简单等特点,近年来被广泛应用于制剂和生物体液中药物的痕量分析。而同步荧光光谱法采用同时扫描激发和发射两个单色器波长的方法,使其不但继承了常规荧光法灵敏度高的优点,同时克服了常规荧光法在分析复杂混合物中遇到光谱重叠和不易分辨的困难,所得图谱更加窄化,避免了瑞利散射和拉曼散射的干扰,在实际样品,如尿样、血样的测定中有效避免了荧光内源性物质的干扰,成为解决多组分荧光物质同时测定的良好手段。与导数技术的联用,增强了对特征光谱精细结构的分辨能力,使得同步荧光法在多组分混合样品的分析中得到广泛应用。
本论文主要探讨了恒波长同步荧光法在药物分析中的应用,全文由综述和研究报告两部分组成。综述部分对同步荧光分析法的特点及分类,恒波长同步荧光法、恒能量同步荧光法、可变角同步荧光法、恒基体同步荧光法的理论基础,以及导数技术的特点进行了评述,主要针对近十年来恒波长同步荧光法在药物分析中的应用进行了总结。研究报告分为同步荧光分析新方法的研究和荧光分析新方法的研究两大部分。第一部分利用同步荧光法避免了尿样中内源性物质的背景干扰,建立了不经分离直接测定尿样中吡罗昔康及氯波必利的新方法;同时基于药物自身荧光,通过选择合适的波长差(Δλ),分别同时测定了混合样品中盐酸氟桂利嗪/盐酸普萘洛尔和地巴唑/西比灵的含量。第二部分基于Ce(Ⅳ)与药物的氧化还原反应,建立了还原性药物美洛昔康和左旋多巴荧光分析的新方法。本研究工作对于药代动力学研究及临床上药物的测定具有一定的应用价值,具体研究内容如下:
1研究了吡罗昔康的荧光特性。实验发现,吡罗昔康自身荧光较弱,但经浓硫酸酸性降解,KMnO_4氧化后具有强荧光,结合一阶导数同步扫描技术,提出了同步荧光法测定尿样中吡罗昔康的新方法。方法线性范围为4.0×10~(-9)～2.4×10~(-6)g/mL,检出限为3.8×10~(-9)g/mL,相对标准偏差为0.54%(n=5, c=1.6×10~(-7)g/mL)。
2研究了氯波必利的荧光特性。实验发现,氯波必利自身具有荧光,甲醛溶液的加入可使其荧光强度显著增强。在pH为6.60的Britton-Robinson(BR)缓冲介质中,以波长差Δλ=30nm进行同步荧光扫描,可消除人体尿液中内源性荧光物质的背景干扰,其同步特征峰的强度与氯波必利的浓度呈线性关系,建立了直接测定尿样中氯波必利的恒波长同步荧光分析方法。氯波必利浓度在2.8×10~(-5)~4.0×10~(-3)g/L范围内与荧光强度呈良好的线性关系,方法检出限为1.1×10~(-5)g/L,相对标准偏差为1.7%(n=11, c=4.0×10~(-5)g/L),方法用于实际尿样中氯波必利含量的直接测定,回收率在95.0~103.8%之间。
3研究了盐酸普萘洛尔和盐酸氟桂利嗪及其混合溶液的同步荧光光谱,结果表明二者的同步荧光谱在Δλ为50nm时得到完全分离,据此建立了同步荧光光谱法同时测定混合样中盐酸普萘洛尔与盐酸氟桂利嗪的新方法。盐酸普萘洛尔与盐酸氟桂利嗪的线性范围分别为1.2×10~(-6)～2.8×10~(-3)g/L和2.0×10~(-5)～3.6×10~(-3)g/L;检出限分别为3.2×10~(-7)g/L和6.8×10~(-6)g/L,用于混合样品中盐酸普萘洛尔与盐酸氟桂利嗪含量的同时测定,回收率在97.6～101.6%之间。
4为探讨同时测定地巴唑及西比灵的含量的可能性,研究了两种药物及其混合液的同步荧光光谱,结果表明在pH=3.29的BR缓冲介质中,波长差为100nm的条件下进行同步荧光扫描,并进行二阶导数处理,可有效消除地巴唑及西比灵彼此间的干扰,分别于278、232nm处测定体系荧光强度,据此建立了二阶导数同步荧光法同时测定混合样中地巴唑与西比灵的新方法。对测定条件进行了详细的研究,地巴唑与西比灵的线性范围分别为1.2×10~(-5)～3.6×10~(-3)g/L和1.2×10~(-4)～6.8×10~(-3)g/L;检出限分别为4.3×10~(-6)g/L和1.0×10~(-4)g/L,用于混合样品中地巴唑与西比灵含量的同时测定,回收率在95.3～103.0%之间。
5研究了美洛昔康-Ce(Ⅳ)-β-环糊精(β-CD)体系的荧光特性。实验发现,美洛昔康自身几乎没有荧光,但其能使Ce(Ⅳ)定量还原为Ce(Ⅲ),Ce(Ⅲ)能发射特征荧光,β-环糊精(β-CD)的加入对体系的荧光强度有增敏增稳作用,基于在λex=260nm和λem=362nm处测定Ce(Ⅲ)的荧光强度,提出了β-CD增敏间接荧光光谱法测定微量美洛昔康的新方法。方法线性范围为8.0×10~(-10)～3.2×10~(-7)g/mL,检出限为3.5×10ˉ10g/mL,相对标准偏差为2.0%(n=5, c=4.0×10~(-8)g/mL)。
6研究了左旋多巴与Ce(Ⅳ)在碱性体系中的荧光特性。实验发现,左旋多巴具有弱的内源性荧光,在pH为9.15的BR缓冲介质中可与Ce(Ⅳ)发生反应,使其荧光强度显著增强,据此提出了测定左旋多巴含量的荧光分析新方法。左旋多巴含量在0.08～3.6μg/mL范围内与其荧光强度线性关系良好,方法检出限为0.018μg/mL。对于片剂中左旋多巴的含量测定,回收率为97.0～100.5%。
The spectrofluorimetric method has been widely used in quantitative analysis because of its highly sensitivity, selectivity and reproducibility as well as its relatively low cost. However, problems of selectivity can occur in multicomponent analysis because of the overlap of the broadband spectra observed. Synchronous fluorescence spectroscopy has been found to have several advantages such as simple spectra, high selectivity and low interference etc. Because of its sharp, narrow spectrum, synchronous fluorescence spectroscopy serves as a very simple, effective method of obtaining data for quantitative determination in a single measurement. In addition, the combination of synchronous fluorimetry and derivatives is more advantageous than conventional spectrofluorimetry in terms of sensitivity, especially when applied to the determination of multi-component analysis.
According to the scanning modes of monochromators, synchronous fluorescence spectroscopy is classified into constant-wavelength, constant-energy, variable-angle and matrix isopotential. At present, the constant-wavelength method is used most extensively.
This thesis consists of a review and two research sections. The review describes the principle, speciality of the normal fluorescence spectra and synchronous fluorescence, and the application of constant-wavelength synchronous fluorescence spectrometry in drugs analysis in recent 10 years. The second section is focused on the study of highly sensitive synchronous fluorescence spectrometry for determination of some medicines. Firstly, in review of normal fluorescence spectra of human urine and many drugs, the spectra are greatly overlapping, such problem was resolved by using synchronous fluorometry. On the basis of this, piroxicam and clebopride have been determined by synchronous fluorometric method which shows good analytical results without any pre-separation steps. Secondly, based on the nature fluorescence of drugs, novel synchronous fluorometric methods have been developed for the simultaneous analysis of propranolol hydrochloride and flunarizine hydrochloride, bendazol and flunarizine hydrochloride, respectively. Finally, meloxicam and levodopa can react with Ce(IV) to produce stong fluorescent species, According to this, new fluorescence methods for determination of meloxicam and levodopa were established.
In conclusion, the proposed methods have the advantages of simplicity, rapidity, precision and sensitivity. The main contents are as follows:
1 A novel synchronous ?uorimetry method for the determination of piroxicam in human urine was described. The experiments indicate that piroxicam has little fluorescence, but the fluorescence intensity of system could be sensitized dramatically by reaction with oxidation after acidic degradation. The results show that piroxicam could be oxidized by potassium permanganate in polyphosphoric acid to yield strong fluorescent species. The combination of synchronous fluorescence atΔλof 70nm and first derivative technique provides good analytical results and permits the direct determination piroxicam in human urine. The fluorescence intensity of system was linearly proportional to the concentration of piroxicam in the concentration range of 4.0×10~(-9)~2.4×10~(-6)g/mL and the detection limit of 3.8×10~(-9)g/mL.
2 A rapid, simple, accurate and selective synchronous ?uorimetry method for the determination of clebopride in human urine was described. The experiments indicate that clebopride has endogenous fluorophore, and the fluorescence intensity was enhanced remarkably when formaldehyde was added into the system. The maximum emission wavelengths of clebopride and the urine blank were at 358.0nm and 414.0nm, respectively. Because the emission spectra of clebopride and fluorescent substance of human urine were overlapped partially, clebopride could not be determined directly by normal fluorimetric method. In order to solve this problem, the synchronous spectrum, maintaining a constant difference ofΔλ=30nm between the emission and excitation wavelengths in Britton-Robinson buffer solution (pH 6.60), was selected as optimum to perform the determination. The background interference of human urine was eliminated effectively. Under the optimum conditions, the fluorescence intensity was linearly proportional to the concentration of clebopride in the range of 2.8×10~(-5)~4.0×10~(-3)g/L. The detection limit was 1.1×10~(-5)g/L. The proposed method has been applied to the determination of clebopride in human urines samples with the recoveries of 95.0~103.8%.
3 A direct method for the simultaneous determination of propranolol hydrochloride and flunarizine hydrochloride using synchronous fluorimetry was described. The maximum emission wavelengths of propranolol hydrochloride and flunarizine hydrochloride were at 354 and 320nm, respectively. Because the emission spectra of two drugs were overlapped partially, propanolol hydrochloride and flunarizine hydrochloride could not be determined directly by normal fluorimetric method. However, the synchronous fluorimetry could be used for determining both drugs simultaneously without separation procedure. The method involved measuring the natural fluorescence of these drugs atΔλ=50nm in the media of acetate buffer solution. The synchronous fluorescence concentration plots were rectilinear over range of 1.2×10~(-6)~2.8×10~(-3)g/L and 2.0×10~(-5)~3.6×10~(-3)g/L for propranolol hydrochloride and flunarizine hydrochloride, respectively with lower detection limits of 3.2×10~(-7) and 6.8×10~(-6)g/L.
4 Simple and sensitive second derivative synchronous fluorometric method was developed for the simultaneous determination of bendazol and flunarizine hydrochloride. The method was based upon measuring the synchronous fluorescence of both the studied drugs in Britton-Robinson buffer of pH 3.29 atΔλ=100nm. The peak amplitude was measured at 278nm (-) and 232nm(+) for bendazol and flunarizine hydrochloride, respectively. With the application of second-order synchronous fluorimetry advantage, the proposed method could be utilized for a simultaneous direct concentration determination of bendazol and flunarizine hydrochloride without pretreatment step. The different experimental parameters affecting the native fluorescence of the studied drugs were carefully studied and optimized. The synchronous fluorescence concentration plots were rectilinear over range of 1.2×10~(-5)～3.6×10~(-3)g/L and 1.2×10~(-4)～6.8×10~(-3)g/L for bendazol and flunarizine hydrochloride, respectively with lower detection limits of 4.3×10~(-6)g/L and 1.0×10~(-4)g/L.
5 A novel and sensitive spectrofluorimetric method for the determination of meloxicam has been developed. The experiments indicated that meloxicam has little fluorescence signal by itself, but it could reduce cerium(IV) to cerium(III). Cerium(III) could emit characteristic fluorescence atλex=260nm andλem=362nm in sulfuric acid medium. Whenβ-cyclodextrin (β-CD) was added into the system, the fluorescence intensity could be enhanced greatly. Based on this meloxicam-cerium(IV)-β-CD fluorescence system, a new fluorescent method was established for the indirect determination of meloxicam. Under optimal experimental conditions, the linear range was 8.0×10~(-10)~3.2×10~(-7)g/mL, the detection limit was 3.5×10ˉ10g/mL. It has been used for the determination of meloxicam in tablets and human urines with satisfactory results.
6 The fluorescence characteristic of levodopa with cerium(IV) in alkalic medium has been studied. The experiments show that levodopa had endogenous fluorophore, but the fluorescence signal was weaker. The fluorescence intensity could be sensitized strongly by the reaction with cerium(IV) in the buffer solution of Britton-Robinson with pH 9.15. Based on this, a new simple, rapid and sensitive fluorescence method for the determination of levodopa has been developed. The linear relationship of fluorescence intensity and levodopa concentration was in the range of 0.08~3.6μg/mL. The detection limit was 0.018μg/mL.
本论文主要探讨了恒波长同步荧光法在药物分析中的应用,全文由综述和研究报告两部分组成。综述部分对同步荧光分析法的特点及分类,恒波长同步荧光法、恒能量同步荧光法、可变角同步荧光法、恒基体同步荧光法的理论基础,以及导数技术的特点进行了评述,主要针对近十年来恒波长同步荧光法在药物分析中的应用进行了总结。研究报告分为同步荧光分析新方法的研究和荧光分析新方法的研究两大部分。第一部分利用同步荧光法避免了尿样中内源性物质的背景干扰,建立了不经分离直接测定尿样中吡罗昔康及氯波必利的新方法;同时基于药物自身荧光,通过选择合适的波长差(Δλ),分别同时测定了混合样品中盐酸氟桂利嗪/盐酸普萘洛尔和地巴唑/西比灵的含量。第二部分基于Ce(Ⅳ)与药物的氧化还原反应,建立了还原性药物美洛昔康和左旋多巴荧光分析的新方法。本研究工作对于药代动力学研究及临床上药物的测定具有一定的应用价值,具体研究内容如下:
1研究了吡罗昔康的荧光特性。实验发现,吡罗昔康自身荧光较弱,但经浓硫酸酸性降解,KMnO_4氧化后具有强荧光,结合一阶导数同步扫描技术,提出了同步荧光法测定尿样中吡罗昔康的新方法。方法线性范围为4.0×10~(-9)～2.4×10~(-6)g/mL,检出限为3.8×10~(-9)g/mL,相对标准偏差为0.54%(n=5, c=1.6×10~(-7)g/mL)。
2研究了氯波必利的荧光特性。实验发现,氯波必利自身具有荧光,甲醛溶液的加入可使其荧光强度显著增强。在pH为6.60的Britton-Robinson(BR)缓冲介质中,以波长差Δλ=30nm进行同步荧光扫描,可消除人体尿液中内源性荧光物质的背景干扰,其同步特征峰的强度与氯波必利的浓度呈线性关系,建立了直接测定尿样中氯波必利的恒波长同步荧光分析方法。氯波必利浓度在2.8×10~(-5)~4.0×10~(-3)g/L范围内与荧光强度呈良好的线性关系,方法检出限为1.1×10~(-5)g/L,相对标准偏差为1.7%(n=11, c=4.0×10~(-5)g/L),方法用于实际尿样中氯波必利含量的直接测定,回收率在95.0~103.8%之间。
3研究了盐酸普萘洛尔和盐酸氟桂利嗪及其混合溶液的同步荧光光谱,结果表明二者的同步荧光谱在Δλ为50nm时得到完全分离,据此建立了同步荧光光谱法同时测定混合样中盐酸普萘洛尔与盐酸氟桂利嗪的新方法。盐酸普萘洛尔与盐酸氟桂利嗪的线性范围分别为1.2×10~(-6)～2.8×10~(-3)g/L和2.0×10~(-5)～3.6×10~(-3)g/L;检出限分别为3.2×10~(-7)g/L和6.8×10~(-6)g/L,用于混合样品中盐酸普萘洛尔与盐酸氟桂利嗪含量的同时测定,回收率在97.6～101.6%之间。
4为探讨同时测定地巴唑及西比灵的含量的可能性,研究了两种药物及其混合液的同步荧光光谱,结果表明在pH=3.29的BR缓冲介质中,波长差为100nm的条件下进行同步荧光扫描,并进行二阶导数处理,可有效消除地巴唑及西比灵彼此间的干扰,分别于278、232nm处测定体系荧光强度,据此建立了二阶导数同步荧光法同时测定混合样中地巴唑与西比灵的新方法。对测定条件进行了详细的研究,地巴唑与西比灵的线性范围分别为1.2×10~(-5)～3.6×10~(-3)g/L和1.2×10~(-4)～6.8×10~(-3)g/L;检出限分别为4.3×10~(-6)g/L和1.0×10~(-4)g/L,用于混合样品中地巴唑与西比灵含量的同时测定,回收率在95.3～103.0%之间。
5研究了美洛昔康-Ce(Ⅳ)-β-环糊精(β-CD)体系的荧光特性。实验发现,美洛昔康自身几乎没有荧光,但其能使Ce(Ⅳ)定量还原为Ce(Ⅲ),Ce(Ⅲ)能发射特征荧光,β-环糊精(β-CD)的加入对体系的荧光强度有增敏增稳作用,基于在λex=260nm和λem=362nm处测定Ce(Ⅲ)的荧光强度,提出了β-CD增敏间接荧光光谱法测定微量美洛昔康的新方法。方法线性范围为8.0×10~(-10)～3.2×10~(-7)g/mL,检出限为3.5×10ˉ10g/mL,相对标准偏差为2.0%(n=5, c=4.0×10~(-8)g/mL)。
6研究了左旋多巴与Ce(Ⅳ)在碱性体系中的荧光特性。实验发现,左旋多巴具有弱的内源性荧光,在pH为9.15的BR缓冲介质中可与Ce(Ⅳ)发生反应,使其荧光强度显著增强,据此提出了测定左旋多巴含量的荧光分析新方法。左旋多巴含量在0.08～3.6μg/mL范围内与其荧光强度线性关系良好,方法检出限为0.018μg/mL。对于片剂中左旋多巴的含量测定,回收率为97.0～100.5%。
The spectrofluorimetric method has been widely used in quantitative analysis because of its highly sensitivity, selectivity and reproducibility as well as its relatively low cost. However, problems of selectivity can occur in multicomponent analysis because of the overlap of the broadband spectra observed. Synchronous fluorescence spectroscopy has been found to have several advantages such as simple spectra, high selectivity and low interference etc. Because of its sharp, narrow spectrum, synchronous fluorescence spectroscopy serves as a very simple, effective method of obtaining data for quantitative determination in a single measurement. In addition, the combination of synchronous fluorimetry and derivatives is more advantageous than conventional spectrofluorimetry in terms of sensitivity, especially when applied to the determination of multi-component analysis.
According to the scanning modes of monochromators, synchronous fluorescence spectroscopy is classified into constant-wavelength, constant-energy, variable-angle and matrix isopotential. At present, the constant-wavelength method is used most extensively.
This thesis consists of a review and two research sections. The review describes the principle, speciality of the normal fluorescence spectra and synchronous fluorescence, and the application of constant-wavelength synchronous fluorescence spectrometry in drugs analysis in recent 10 years. The second section is focused on the study of highly sensitive synchronous fluorescence spectrometry for determination of some medicines. Firstly, in review of normal fluorescence spectra of human urine and many drugs, the spectra are greatly overlapping, such problem was resolved by using synchronous fluorometry. On the basis of this, piroxicam and clebopride have been determined by synchronous fluorometric method which shows good analytical results without any pre-separation steps. Secondly, based on the nature fluorescence of drugs, novel synchronous fluorometric methods have been developed for the simultaneous analysis of propranolol hydrochloride and flunarizine hydrochloride, bendazol and flunarizine hydrochloride, respectively. Finally, meloxicam and levodopa can react with Ce(IV) to produce stong fluorescent species, According to this, new fluorescence methods for determination of meloxicam and levodopa were established.
In conclusion, the proposed methods have the advantages of simplicity, rapidity, precision and sensitivity. The main contents are as follows:
1 A novel synchronous ?uorimetry method for the determination of piroxicam in human urine was described. The experiments indicate that piroxicam has little fluorescence, but the fluorescence intensity of system could be sensitized dramatically by reaction with oxidation after acidic degradation. The results show that piroxicam could be oxidized by potassium permanganate in polyphosphoric acid to yield strong fluorescent species. The combination of synchronous fluorescence atΔλof 70nm and first derivative technique provides good analytical results and permits the direct determination piroxicam in human urine. The fluorescence intensity of system was linearly proportional to the concentration of piroxicam in the concentration range of 4.0×10~(-9)~2.4×10~(-6)g/mL and the detection limit of 3.8×10~(-9)g/mL.
2 A rapid, simple, accurate and selective synchronous ?uorimetry method for the determination of clebopride in human urine was described. The experiments indicate that clebopride has endogenous fluorophore, and the fluorescence intensity was enhanced remarkably when formaldehyde was added into the system. The maximum emission wavelengths of clebopride and the urine blank were at 358.0nm and 414.0nm, respectively. Because the emission spectra of clebopride and fluorescent substance of human urine were overlapped partially, clebopride could not be determined directly by normal fluorimetric method. In order to solve this problem, the synchronous spectrum, maintaining a constant difference ofΔλ=30nm between the emission and excitation wavelengths in Britton-Robinson buffer solution (pH 6.60), was selected as optimum to perform the determination. The background interference of human urine was eliminated effectively. Under the optimum conditions, the fluorescence intensity was linearly proportional to the concentration of clebopride in the range of 2.8×10~(-5)~4.0×10~(-3)g/L. The detection limit was 1.1×10~(-5)g/L. The proposed method has been applied to the determination of clebopride in human urines samples with the recoveries of 95.0~103.8%.
3 A direct method for the simultaneous determination of propranolol hydrochloride and flunarizine hydrochloride using synchronous fluorimetry was described. The maximum emission wavelengths of propranolol hydrochloride and flunarizine hydrochloride were at 354 and 320nm, respectively. Because the emission spectra of two drugs were overlapped partially, propanolol hydrochloride and flunarizine hydrochloride could not be determined directly by normal fluorimetric method. However, the synchronous fluorimetry could be used for determining both drugs simultaneously without separation procedure. The method involved measuring the natural fluorescence of these drugs atΔλ=50nm in the media of acetate buffer solution. The synchronous fluorescence concentration plots were rectilinear over range of 1.2×10~(-6)~2.8×10~(-3)g/L and 2.0×10~(-5)~3.6×10~(-3)g/L for propranolol hydrochloride and flunarizine hydrochloride, respectively with lower detection limits of 3.2×10~(-7) and 6.8×10~(-6)g/L.
4 Simple and sensitive second derivative synchronous fluorometric method was developed for the simultaneous determination of bendazol and flunarizine hydrochloride. The method was based upon measuring the synchronous fluorescence of both the studied drugs in Britton-Robinson buffer of pH 3.29 atΔλ=100nm. The peak amplitude was measured at 278nm (-) and 232nm(+) for bendazol and flunarizine hydrochloride, respectively. With the application of second-order synchronous fluorimetry advantage, the proposed method could be utilized for a simultaneous direct concentration determination of bendazol and flunarizine hydrochloride without pretreatment step. The different experimental parameters affecting the native fluorescence of the studied drugs were carefully studied and optimized. The synchronous fluorescence concentration plots were rectilinear over range of 1.2×10~(-5)～3.6×10~(-3)g/L and 1.2×10~(-4)～6.8×10~(-3)g/L for bendazol and flunarizine hydrochloride, respectively with lower detection limits of 4.3×10~(-6)g/L and 1.0×10~(-4)g/L.
5 A novel and sensitive spectrofluorimetric method for the determination of meloxicam has been developed. The experiments indicated that meloxicam has little fluorescence signal by itself, but it could reduce cerium(IV) to cerium(III). Cerium(III) could emit characteristic fluorescence atλex=260nm andλem=362nm in sulfuric acid medium. Whenβ-cyclodextrin (β-CD) was added into the system, the fluorescence intensity could be enhanced greatly. Based on this meloxicam-cerium(IV)-β-CD fluorescence system, a new fluorescent method was established for the indirect determination of meloxicam. Under optimal experimental conditions, the linear range was 8.0×10~(-10)~3.2×10~(-7)g/mL, the detection limit was 3.5×10ˉ10g/mL. It has been used for the determination of meloxicam in tablets and human urines with satisfactory results.
6 The fluorescence characteristic of levodopa with cerium(IV) in alkalic medium has been studied. The experiments show that levodopa had endogenous fluorophore, but the fluorescence signal was weaker. The fluorescence intensity could be sensitized strongly by the reaction with cerium(IV) in the buffer solution of Britton-Robinson with pH 9.15. Based on this, a new simple, rapid and sensitive fluorescence method for the determination of levodopa has been developed. The linear relationship of fluorescence intensity and levodopa concentration was in the range of 0.08~3.6μg/mL. The detection limit was 0.018μg/mL.
引文
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