Sun Hanwen, Li Lixin, Suo Ran, Qiao Fengxia, Liang Shuxuan
(Key Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding, 071002, China)

Received Sep. 23, 2003; Supported by the Natural Science Foundation of Hebei Province (203110).

Abstract The speciation of Zn in Chinese herbal medicines was developed by vapour generation-atomic fluorescence spectrometry with nickel combined with 1,10-phenanthroline as an enhancement reagent. The effects of measurement conditions and chemical parameters on Zn fluorescence signal were investigated and optimized. A n-octanol-water system was used to study the distribution of Zn in herb decoctions under the stomach and intestine acidity. The speciation and content of Zn in herbal medicines and their water decoctions were related with the component of the medicine, acidity of target. The concentrations of the total Zn, water-soluble Zn and n-octanol-soluble Zn in seven Chinese herbal medicines under stomach and intestine acidity were analyzed by the proposed method with satisfactory recovery.
Keywords Vapour generation Atomic fluorescence spectrometry; Chinese herbal medicines; Speciation; Zinc

1 INTRODUCTION
Vapour generation-Atomic absorption spectrometry (VG-AAS) has been applied to the determination of hydride-formed elements, such as Se, Te, As, Sb, Bi, Pb, Ge, Sn, Hg etc, using quartz tube atomizer or graphite furnace atomizer with in situ trapping preconcentration^[1]. In recent years, efforts have been made for developing new reaction systems to determine more elements, e.g. cadmium^[2] and copper^[3]. Xu et al. investigated enhancement reagents for response signals of copper, gold and thallium in flow injection vapor generation atomic absorption spectrometry^[4]. A new method was described for the direct determination of As and Sb in water by hydride generation-derivative atomic absorption spectrometry^[5].
    Vapour generation atomic fluorescence spectrometry (VG-AFS) is a highly sensitive and effective method. It has been applied to the determination of selenium and tellurium^[6], lead and Hg^[7], cadmium and arsenic^[8] in biological samples. Guo^[9] had studied the reaction of Zn in aqueous solution with sodium tetrahydroborate and first found the volatile species generation of Zn. However, the nature of such species has not been investigated in detail and the generation efficiency was relatively poor. A new method was proposed in our previous paper for the determination of Zn in food by atomic fluorescence spectrometry with vapour generation from surfactant-based organized media. The presence of cetyltrimethylammonium bromide(CTAB) could, both thermodynamically and kinetically, facilitate the generation of volatile species of Zn. The advantages of vapour generation from the CTAB media were contrasted with that from aqueous media in sensitivity and precision of the Zn determination^[10].
    Chinese herbal medicines are some of the oldest alternative and complementary medicines and their ever-increasing use is a good indication of the public interest in such medicines. They have long been used in Chinese traditional healing systems and their pharmacology associated with the inorganic elements. Zn is an important element and has essential effects on human health. There are over eighty kinds of Zn-contained enzyme, the activity of over two hundreds enzymes is related with Zn element^[11]. The action of trace Zn in Chinese herbal medicine on human body is depended on speciation of Zn. The determination of total Zn and speciation of Zn in Chinese herbal medicines by vapour generation atomic fluorescence spectrometry have not been reported up to now. Therefore, it is important to develop a new extraction method and speciation analysis of Zn for study of pharmacology.
    The main purpose of this paper is to select a new enhancement reagent for Zn response signals and develop a new method for the determinations of total Zn, water-soluble Zn and n-octanol-soluble Zn in medicines by vapour generation-atomic fluorescence spectrometry.

2 EXPERIMENTAL
2.1 Apparatus
A AFS-230 double channel none-dispersive atomic fluorescene spectrometer (Beijing Haiguang Instrument Company,China) was used and controlled through a computer. The light source, coded hollow cathode lamp, was operated in a double-modulated pulsed mode, which offers improved stability, high radiation intensity and a longer lifetime.
    An intermittent flow vapor generation system was employed throughout this work. The recommended operating conditions are given in Table 1.

Table 1. The operating parameters of the AFS

2.2 Reagents
Analytical grade reagents and de-ionized water were used throughout the study. A 0.3-1% (m/v) solution of potassium tetrahydroborate was prepared daily in de-ionized water containing sodium hydroxide (5g/L) to keep the reductant stable.
    A 0.4% (m/v) of 1,10-phenanthroline (Shenyang Chemical Co., Shenyang, China) was prepared by dissolving 0.2 g of 1,10-phenanthroline in 50mL of de-ionized water.
    A stock solution of Zn, 1000 mg/mL, was prepared by dissolving 0.1000g of pure Zn in a 5 ml of 6 mol L^-1 hydrochloric acid and diluting to 100 mL with de-ionized water. The working solutions were prepared by diluting appropriate aliquots from the stock solution.
2.3 Procedure
Vapour generation was carried out using an intermination flow program and the operating conditions presented in Table 2. All measurements were performed under the recommended conditions.

Table 2 Intermittent flow program and operating conditions

^aRotations per minute

    A home-made programmable intermittent flow reactor was used throughout the work. The configuration of the device was similar to that of a continuous flow reactor, but the operation of the pump could be programmed in several steps for each measurement. At every step the rotation rate and time could be programmed by the operator. In this work, the operation of the pump during each measurement consisted of three steps. At the first step, the sampling tube was placed in the test solution, the sample was propelled by the pump at 5.8 mL/ min and the potassium tetrahydroborate solution at 10.6 mL/ min for 10s. At the second step, the pump was stopped for 4s thus allowing the sampling tube to be changed over to the carrier solution. At this stage, the sample stayed in the storage coil which was in front of the mixing joint of the manifold and consequently no reaction occurred between the sample solution and the reductant. At the third step, the pump rate was raised to 100 rpm for 16s, the carrier solution was propelled at 11.2 mL/ min, and the potassium tetrahydroborate solution at 16.9 mL/ min, rapidly pushing the sample and the reductant into the mixing coil and gas-liquid generator. At this stage, volatile Zn species were formed, transported to the quartz furnace and atomized therein. The signal was recorded and displayed on the screen. After the third step, the pump was stopped again and made ready for the next determination.
2.4 Sample treatment
2.4.1 Analysis of Zn in Chinese herbal medicine
Each herb sample of 1.000g was digested in a 50 mL beaker on an electric hot-plate for 1 h with 8 mL of nitric acid and 2 mL of perchloric acid. Then, each digest solution was gently heated, and any excess acid was removed and 2 mL of nitric acid was added into each sample, and the digest solution was heated again until a clear solution was obtained. The digest solution along with the digest solution which was obtained by dissolving residues with 1% nitric acid, were transferred into a 50 mL volumetric flask and diluted to the volume with de-ionized water for the determination of total Zn in Chinese herbal medicines. The sample blank was treated in the same way.
2.4.2 Analysis of total water-soluble Zn in water decoctions
A 20.00 g of a Chinese herbal medicine sample in a 400mL beaker was added triplet aliquots of de-ionized water ( 200 mL, each) and heated on an electric hot-plate for slight boiling of 30 min. The obtained decoctions were filtered by a 0.45mm membrane. The filtrate was concentrated to obtain about 100 mL water decoction for analysis.
    An aliquot of the water decoction (30.0 ml) was used for the determination of total water-soluble Zn. The 30.0 ml solution was heated gently until about 5 ml of solution was obtained, then 8 ml of nitric acid and 2 ml of perchloric was added and gently heated on a hot plate to dryness. After cooling, the residue was dissolved with 1% (v/v) nitric acid and diluted to 25 ml of volume with de-ionized water for determining the content of total water-soluble Zn by VG-AFS.
2.4.3 Speciation of Zn in water decoctions at stomach and intestine acidity
Two aliquots of the above water decoction (30.0 mL, each) were added into two beakers, respectively. One was adjusted to the acidity of stomach (pH=1.3), the other was adjusted to the acidity of intestines (pH=7.6) with 2.0 mol L^-1 hydrochloric acid and 30% v/v NH₃^.H₂O. After over a night, they were transferred into a separatory funnel, added 10.0 mL n-octanol solution, respectively, and extracted with oscillating for 2 h, The water phase samples were digested ditto as water-boiling sample. At last the residues were diluted to 25 ml in volumetric flasks with 1% (v/v) nitric acid for the determination of water-soluble Zn in the water decoction. Then concentrations of n-octanol-soluble Zn were obtained by subtracting the concentrations of water-soluble Zn from the concentrations of total water-soluble Zn in the water decoctions, respectively.

3 RESULTS AND DISCUSSIONS
3.1 Effects of atomizer temperature and solution temperature
An influence of the atomizer temperature in the range of 200 -700ºC on the Zn signals was studied. The signal intensity decreased and the noise levels increased when the furnace temperature at higher than 500ºC because the vapor expansion and the furnace radiation were increased at a higher temperature. 400ºC of the atomizer temperature can provide enough energy to atomize the volatile Zn and was used for atomizing in all the experiments.
    The Zn signal for 30 ng/mL Zn solution was monitored at different solution temperatures. No signal could be observed when the solution temperature was below 14ºC. The signal increased drastically with raising temperature from 15ºC to 20ºC, and stabilized when the temperature was beyond 20ºC. All the experiments were carried out at the zoom temperature (above 20ºC).
3.2 Effects of carrier gas and shield gas flow rate
Pure argon was used as both the carrier gas and the shield gas. The argon flow was used to transfer generated hydride from the hydride generator to the atomizer quarts cell. The flow rate and flow stability of carrier gas usually had significant effect on the sensitivity and repeatability of the method. Various flow rates, which influenced the sensitivity and stability of the instrument, were tested in this study. If the carrier gas was at too low flow rate, it could not quickly sweep the vapor of the analyte into the inner tube of a quartz furnace, and if it was at too high flow rate, the analyte would be diluted, and the carrier gas could introduce into the furnace tube and reduce the residence times of the analyte in atomizer quarts cell by the carrier gas. The shield gas was used to prevent extraneous air from entering the flame. Experimental results showed that the Zn fluorescence intensity increased up to reach a maximum value when the carrier gas(Ar) flow and the shield gas(Ar) flow were 300ml min^-1 and 700 ml min^-1, respectively. A 300ml min^-1 for carrier gas and a 700 ml min^-1 for shield gas were chosen as the optimum argon flow rate for the determination of Zn in this study.
3.3 Effect of observation height
The observation height was the distance from the quartz furnace outlet to the point where the atomic fluorescence signal was measured. The observation height was evaluated in the range of 8 to 15 mm. The signal intensity increased drastically with the increase of observation height in the range of 8 to 12 mm. However, a too high observation height would reduce the signals because of oxidation of the analyte by the oxygen in air from entering the flame. In this study an observation height of 13 mm was used.
3.4 Effect of KBH₄ concentration
KBH₄ was used as both a reducer and a hydrogen supplier, which was necessary to sustain the argon-hydrogen flame. The variation of the Zn fluorescence intensity with KBH₄ concentration in the range of 0.2 to 3%(w/v) was investigated. The fluorescence intensity increased drastically with raising KBH₄ concentration from 0.2% to 2%(w/v), and decreased when beyond 2% of the KBH₄ concentration. In this work, a 2% (w/v) of KBH₄ concentration in 0.5%(w/v) NaOH solution, which provided a good signal-to-noise ratio, was employed for vapour generation.
3.5 Effect of acid medium and reacting acidity          
Generation efficiency of Zn volatile species depended on the acidity of reaction medium and the acid species strongly. The effects of hydrochloric acid, nitric acid, phosphoric acid and sulfuric acid acidity from 0.02mol L^-1 to 0.4 mol L^-1 on the efficiency of vapour generation were investigated with 2.0% (w/v) KBH₄ as a reducer. Experimental results were shown in Fig.1. The significant decrease of Zn fluorescence intensity was observed for phosphoric acid and sulfuric acid medium, so it might not be the best choice.　For hydrochloric acid and nitric acid, low-acidity condition was benefit for producing higher fluorescence intensity. Using nitric acid as sample medium, a plateau was existed in the nitric acid concentration from 0.1mol L^-1 to 0.15 mol L^-1. The high nitric acid concentration has negative effect for the determination due to the dilution effect of large account of hydrogen generated in the reaction process. However, using hydrochloric acid as medium, a plateau was existed in the acidity range 0.08mol L^-1 to 0.15 mol L^-1, and the reduction of sensitivity was observed when the concentration of hydrochloric acid up to or beyond 0.15 mol L^-1.
    From the results on the effect of sample solution's acidity on the fluorescence intensity of 70ng Zn, 0.1mol/L hydrochloric acid was chosen as sample solution acidity in this study.

Fig.1 The effect of medium acidity on the fluorescence intensity of 70ng Zn

    At the same time, the influence of HCl concentration as carrier liquid on fluorescence signals was investigated. The results showed that the HCl concentration in the range of 0.05-0.3mol/L had a significant effect on the measurement of Zn. 0.1mol/L of HCl was used for the carrier liquid medium throughout this work.
3.6 Selection of enhancement reagent             
For the VGAAS determination of Zn, it was more important to choose an appropriate enhancement reagent for enhancing the vapor generation efficiency of Zn using quartz tube as atomizer because the sensitivity of the VGAAS system without enhancement reagent was too low. The sodium diethanlydithio-carbonate (DDTC), 1, 10-phenanthroline, and palladium were used as enhancement reagents for VGAAS, respectively. DDTC, 1,10-phenanthroline, and palladium combined with rodamine showed the enhanced effect on gold, respectively, and the sensitivities were increased by 1-2 magnitude^[4]. In order to choose an appropriate enhancement reagent for enhancing the Zn fluorescence signal, 1,10-phenanthroline, citric acid, oxalic acid, tartaric acid, thiourea and nickel were investigated as enhancement reagent, respectively. It was found that nickel, 1,10-phenanthroline, and nickel combined with 1,10-phenanthroline could improve the Zn signal with a different degree, The results were shown in Fig. 2.

Fig. 2 The effect of enhancement reagent concentration on the fluorescence intensity of 100ng Zn.
B: nickel(mg/L), D: nickel( mg/L) combined with 1,10-phenanthroline (10^-2mg/L) E: 1,10-phenanthroline(10^-2mg/L)

    Using nickel combined with 1,10-phenanthroline as enhancement reagent, the Zn fluorescence intensity would be very higher than that with nickel or 1,10-phenanthroline as respective enhancement reagent. From Fig.2, it was clear that Zn signal was increased greatly and reached a wide plateau with nickel (0.2-2.0mg/L) combined with 1,10-phenanthroline (2-20mg/L) as enhancement reagent, which was easy to control and especially convenient for analysis. So 1.0 mgL^-1 of nickel combined with 12mg/L of 1,10-phenanthroline were kept constant in sample solution as the optimum concentration for the work.
3.7 Interference studies
Using the recommended conditions, the interferences from coexisting ions on the determination of 40mg/L Zn were investigated. The effects of the common hydride-forming elements seem to be less troublesome for Zn determinations. Up to 40mg/L of Mg, 24 mg/L of Ca and Ba, 20 mg/L of Mn, 12 mg/L of Cr and Sr, 8 mg /L of As, Se and Ge, 4 mg/L of Te, Fe, Sn and Cd , and 2 mg/L of Sb would not disturb the determinations of Zn.
3.8 Analytical performance
Under optimum experimental conditions, linearity relation between fluorescence peak area intensity and Zn concentrations in the range of 5-80mg/L could be described by regression equation: I_f = 0.042C+8.14, with a regression coefficient of 0.999. The detection limit(3s) was 0.78 mg/L , The recovery was a range of 97.5% to 100.3% with relative standard deviations of 0.22%.
3.9 Analysis of herbal medicines samples
3.9.1 Analysis of total Zn and water-soluble Zn in herbal medicines
The contents of total Zn in seven Chinese herbal medicines and their decoctions were determined by vapour generation-atomic fluorescence spectrometry under optimum experimental conditions. In order to compare the water-dissolving capability of Zn in seven Chinese herbal medicines, the content of water-soluble Zn in Chinese herbal medicines and the ratios of water-soluble Zn content to total Zn content in Chinese herbal medicine were calculated. The results, along with recoveries were given in Table 3.

Table 3 Content of total Zn and water-soluble Zn in herbal medicines

    For the studied seven medicines, the total Zn contents were in the range of 23.05-126.78 mg/g with recovery of 97.5-101.1%. The total Zn content in Radix Notoginseng was the highest. The dissolvable ratio of Zn for Radix Isatidis was much higher than that for Discorea nipponica Makino and the other herbs. There was no relation between the total Zn content and the dissolvable ratio for the seven medicines. At the different chemistry and physical environments, the structures of every species were different, so the distribution of Zn species in water decoctions was not similar to the herbs. The water-dissolving capability of Zn was related with the kind of medicine and the speciation of analyte in the medicine. Therefore, only using the total concentrations of Zn was unreasonable to evaluate the action of Chinese herbal medicine, and the action of Zn in water decoction was a key provenance for evaluation of medicine effect.
3.9.2 Speciation of water-soluble and n-octanol-soluble Zn at stomach and intestine acidity
The water decoction medicine was the really one for people to treat diseases, and the quantum of Zn, especially their species, in water decoction was the actual contribution to the therapy. An n-octanol system was used to study the distribution of Zn in water decoction under the simulation acidity in our stomach and intestine^[12]. The distribution of Zn speciation in water decoctions was expressed with equation k_ow=c_o/c_w, where c_o was the concentration of n-octanol-soluble Zn, and c_w was the concentration of water-soluble Zn. Analytical results of water-soluble Zn and n-octanol-soluble Zn in decoctions under gastric and intestinal acidity were given in Table 4.

Table 4 Analytical results of water-soluble Zn and n-octanol-soluble Zn in decoctions under gastric and intestinal acidity

    The total water-soluble Zn and n-octanol-soluble Zn of Radix Notoginseng, were the highest in the seven decoctions under gastric and intestinal acidity. Under intestinal acidity K_ow (0.58) of Radix Notoginseng was higher than that of the others. For Radix salviae miltiorrhizae, K_ow (0.86) under gastric acidity is the highest, and K_ow (0.44) under intestinal acidity was the higher, too. The water-dissolving capability of Zn was related with the kind of medicine herb. The acidity of the decoction had obvious effects on the distribution of water-soluble Zn and n-octanol-soluble Zn. For Radix Glycyrrhizae, Radix Notoginseng, Rhizoma anemarrhenae, Fructus Ligustri Lucidi and Radix salviae miltiorrhizae, the K_ow of Zn under gastric acidity was higher than that under intestinal acidity for oneself. and for Discorea nipponica Makino and Radix Isatidis, it was reverse. It was shown that Zn would be removed from water-soluble form to n-octanol-soluble form with increasing or decreasing pH value of water decoction. If the K_ow of Zn under gastric acidity was lower than that under intestinal acidity, the water-soluble Zn would be translated into n-octanol-soluble Zn with the increasing pH. and if it was reverse , the n-octanol-soluble Zn would be translated into water-soluble Zn with the increasing PH. Therefore, medicine effects of water decoction would not be evaluated only by the total content of a trace element in water decoction while studying pharmacology action of herbal medicine. It was indicated that the concentration of water-soluble and n-octanol-soluble species and its ratios were related with the kind of medicine and the acidity of the decoction. In a word, the component of medicines, acting target, target acidity and medicine compatibility should be taken into account when the effect of herbal medicine was studied.

4 CONCLUSIONS
Nickel combined with 1,10-phenanthroline was an effective enhancement reagent for vapor generation atomic fluorescence spectrometry. The present method could be applied for speciation analysis of total Zn, water-soluble Zn and n-octanol-soluble Zn in decoctions with higher sensitivity. The distribution of water-soluble Zn and n-octanol-soluble Zn in decoction was related with the component of medicine and acidity of the decoction. The component of medicine and acidity of target and compatibility of medicines had great effects on the speciation and concentration of Zn in their decoctions.

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