Investigation of Distribution for Trace Lead and Cadmium in Chinese Herbal Medicines and Their Decoctions by Graphite Furnace Atomic Absorption Spectrometry

doi:10.4236/ajac.2011.22026

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American Journal of Anal yt ical Chemistry, 2011, 2, 217-222

doi:10.4236/ajac.2011.22026 Published Online May 2011 (http://www.SciRP.org/journal/ajac)

Investigation of Distribution for Trace Lead and Cadmium

in Chinese Herbal Medicines and Their Decoctions by

Graphite Furnace Atomic Absorption Spectrometry

Hanwen Sun, Lixin Li

Key Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and

Environmental Science, Hebei University, Baoding, China

E-mail: [email protected]

Received February 16, 2011; revised March 7, 2011; accepted March 15, 2011

Abstract

Lead and cadmium in herbal medicines are highly toxic to living organisms even in low concentrations. An

effective method is developed for analysis of trace lead and cadmium in Chinese herbal medicines and their

decoctions by graphite furnace atomic absorption spectrometry (GFAAS). The effects of analytical condi-

tions on absorbance were investigated and optimized. A water-dissolving capability for Pb and Cd was in-

vestigated, and the contents of different species in five Chinese herbal medicines and their decoctions were

analyzed. The content ratios (kow) of n-octanol-soluble Pb or Cd to water-soluble Pb or Cd were evaluated,

and the distribution of Pb and Cd in water decoction at stomach and intestine acidities was developed, in the

first time. The contents of water-soluble Pb and Cd, n-octanol-soluble Pb and Cd, and their content ratios

were related with the kind of medicine and the acidity of the decoction. The proposed method has the advan-

tages of simple operation, high sensitivity and high speed, with 3 σ detection limits of 4.2 pg for Pb and 0.1

pg for Cd.

Keywords: Distribution, Lead, Cadmium, Herbal Medicines, Decoctions, Graphite Furnace Atomic

Absorption Spectrometry

1. Introduction

Chinese herbal medicine has been used as a form of

treatment for a multitude of ailments ranging from acute

infections to chronic fatigue. Study on the trace elements

in Chinese herbal medicine can provide science provi-

dence for pharmacology and toxicology. Lead has well-

documented toxic effects on plants, animals, and micro-

organisms. Cadmium exposure can produce a wide vari-

ety of acute and chronic effects in humans, leading to a

build-up of cadmium in the kidneys that can cause kidney

disease. Some cases of intoxication with Pb and Cd re-

sulting from the ingestion of Chinese herbal medicines

have been reported [1,2]. Therefore, the analysis of trace

Pb and Cd in herbal medicines and their decoctions is

very important for pharmacological and toxicological

evaluations.

A series of atomic spectrometric methods were re-

ported for the determination of Pb and/or Cd in herbal

medicines. Atomic absorption spectrometry is the ana-

lytical technique, most employed for metal analysis due

to its low interference level and reasonable sensitivity. A

pre-concentration flow injection analysis–flame atomic

absorption spectrometry (FAAS) method was reported

for determination of Pb content in medicinal plants with

the limit of detection (LOD) of 19 µg·L–1 [3]. A solid

sampling–FAAS method was used for determination of

Cd and Pb in medicinal plants, relative standard devia-

tions (RSDs) were below 10% (n = 5) and LODs were

0.1 µg·kg–1 and 12 µg·kg–1 for Pb and Cd, respectively

[4]. To increase method sensitivity graphite furnace

atomic absorption spectrometry (GFAAS) was frequently

used to determine heavy metals for the quality control in

herbal medicines. Several GFAAS methods had been

reported with higher sensitivity, such as for the determi-

nation of Pb in Chinese herbal medicine with the LOD of

0.1 μg·kg–1[5], and for the determination of Pb and Cd in

Chinese crude drugs with the LOD of 11.6 pg and 2 pg

[6], and 6 μg·kg–1 and 0.208 μg·kg–1 [7] for Pb and Cd,

respectively. GFAAS was utilized to the determination

H. W. SUN ET AL.

218

of 4 metal element in 13 herbs of tocolysis formulation

with the LOD of 0.45 ppb (μg·L–1) for Pb and 0.03 ppb

(μg·L–1) for Cd [8], and to the quality control in Argen-

tinian herbal medicines with the LOD of 0.06μg·L–1 for

Pb and 0.008 μg·L–1 for Cd [9]. GFAAS and inductively

coupled plasma mass spectrometry (ICP-MS) had been

used for the determination of arsenic, cadmium and lead

for quality control monitoring purposes of Bulgarian

herbs and their infusions, and the LODs using GFAAS

were 0.5 μg·L–1 for Pb and 0.01 μg·L–1 for Cd [10]. Hy-

dride generation-atomic fluorescence spectrometry

(HGAFS) method was described for simultaneous de-

termination of trace Cd and arsenic in biological samples

with the LOD of 0.01 μg·L–1 for Cd [11]. Otherwise,

a–reversed-phase high-performance liquid chromatogra-

phy (RP-HPLC) method, was developed for the simulta-

neous determination of heavy metal ions in Chinese

herbal medicine. The method had low LOD value of

0.004 μg·L–1 for Pb and 0.003 μg·L–1 for Cd, but analyti-

cal procedure was complicated, and required pre-column

derivatized with tetra-(4-chlorophenyl)-porphyrin (T4-

CPP) to form the colored chelates [12]. All the methods

reported above were used for effective determination of

total content of analyte. However, because the action of

trace elements in Chinese herbal medicine on human

body is mostly depended on the combination forms of

elements, so the distribution analysis of trace elements is

more important than total content analysis.

The n-octanol system was used to study the distribu-

tion of major-element or micro-element in water decoc-

tion in our stomach and intestine, and to appraise the

medicine affection in pharmacology [13]. The combina-

tion forms of Zn and Mn in some Chinese herbal medi-

cines was investigated by FAAS using n-octanol system,

but since FAAS normally allows the quantification of

elements only at µg·g–1 levels [14,15]. A new method

was developed for simultaneous determination of trace

arsenic and antimony in Chinese herbal medicines by

HGAFS with a Soxhlet extraction system and n-octanol-

water extraction system [16]. Lead and cadmium in her-

bal medicines are highly toxic to living organisms, how-

ever there was no report for analysis of trace Pb and Cd

in Chinese herbal medicines and their decoctions up to

now.

The main purpose of our work is to study the distribu-

tion of Pb and Cd at stomach and intestine acidities.

Chemical modification technique was used for GFAAS

to increase the analytical sensitivity and to overcome

matrix interference. The n-octanol-water extraction sys-

tem was used for the analysis of trace Pb and Cd in the

decoctions of Chinese herbal medicines. The content of

water-soluble and n-octanol-soluble analyte and its ratio

were investigated. The proposed method was used to ex-

amine the distribution of Pb and Cd at stomach and in-

testine acidities. The method was used for the determina-

tion of total Pb/Cd, water-soluble and n-octanol-soluble

Pb/Cd in Chinese herbal medicines and their decoctions

with satisfactory results.

2. Materials and Methods

2.1. Instrumentation

A PE-1100B graphite furnace atomic absorption spec-

trometer (Perkin Elmer Co.) equipped with Pb and Cd

hollow cathode lamps (Beijing Haiguang Instrument Co.,

China) was used for all the measurements. Pyrocoated

graphite tubes were used. The instrument operating pa-

rameters are given in Table 1.

2.2. Reagents and Samples

All reagents were of analytical-reagent grade except

when indicated otherwise. De-ionized water was used

throughout this work. The working solutions of Pb and

Table 1. The operating parameters of GFAAS.

Parameters Pb Cd

Measurement wavelength/nm 283.3 228.8

Lamp current/mA 6 6

Slit/nm 0.7 0.7

Charring temperature/˚C 60 120 60 120

Ramp time/s 5 10 5 10

Hold time/s 10 10 10 10

Pyrolysis temperature/˚C 800 800

Ramp time/s 10 15

Hold time/s 10 15

Atomization temperature/˚C 1700 1800

Ramp time/s 0 0

Hold time/s 5 4

Cleaning temperature/˚C 2500 2500

Ramp time/s 1 1

Hold time/s 3 3

Background correction D-lamp D-lamp

Measurement model Peak area Peak area

Integral time/s 3 3

Carries gas flow/mL·min–1 300 300

H. W. SUN ET AL.219

Cd were prepared by diluting appropriate aliquots from

the stock solution (1 mg·mL–1, provided by the National

Steel Material Testing Center, China). Mg(NO3)2,

NH4NO3, NH4H2PO4 and PdCl2 were used to study modi-

fication effects. A n-octanol solution was used for analy-

sis.

Samples of Chinese herbal medicines, Common ane-

marrhena rhizome, Liquorice, Dioscorea nipponica ma-

kino, Radix salviae miltiorrhizae and Fructus ligustri

lucidi, were bought from a market.

2.3. Procedure

For the analysis of total Pb/Cd in herbal medicines, com-

plete digestion of a herbal sample was performed by wet

digestion with a mixture of nitric acid and hydrochloric

acid. A 1.000 g sample of Chinese herbal medicine was

digested with 8 mL of nitric acid and 2 mL of perchloric

acid in a beaker covered with a watch glass on an electric

hot-plate for about one hour, then each digest solution

was gently heated, and any excess acid was removed.

After cooling to room temperature (25˚C), the residues

were dissolved with 1% nitric acid. The content was

transferred to a volumetric flask and diluted to 50 mL

with 1% nitric acid for the determination of total Pb/Cd.

The sample blank preparation and recovery test were

carried out in the same way.

For the analysis of total water-soluble Pb/Cd in water

decoctions, a 20.00 g sample of Chinese herbal medi-

cines in a 400 mL beaker was extracted with triplet de-

ionized water (200 mL, each) and heated on an electric

hot-plate to slight boiling of 1 h. The obtained decoctions

were filtered with a 0.45 µm membrane. The water de-

coction was obtained by concentrating the filtrate to 100

mL. An aliquot of the water decoction (30.0 mL) was

used for the determination of total water-soluble Pb/Cd.

The 30.0 mL solution was heated until about 5 mL of

solution was obtained, then 8 mL of nitric acid and 2 mL

of perchloric acid were 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 the analysis of Pb and Cd 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

the stomach (pH 1.3) with 2.0 mol/L hydrochloric acid

and 30% (v/v) NH3·H2O, the other was adjusted to the

acidity of intestine (pH 7.6). They were translated into a

separatory funnel, and then 10.0 mL of n-octanol was

added, oscillating for extraction for 2 h. The water phase

obtained was diluted to 25.0 mL with 1% (v/v) nitric acid

for the determination of water-soluble Pb/Cd in the water

decoctions. Then contents of n-octanol-soluble Pb/Cd

were obtained by subtracting the content of water-soluble

Pb/Cd from that of total Pb/Cd in the water decoctions,

respectively.

3. Results and Discussion

3.1. Effect of Acid Medium and Acidity

The effects of hydrochloric acid, nitric acid, phosphoric

acid and sulfuric acid acidity from 1% to 2% (v/v) on the

absorbance of Pb and Cd were investigated. Used hy-

drochloric acid or phosphoric acid as a medium, the back-

ground interferences would be increased, and the ab-

sorbance would be lower than using sulfuric acid as a

medium. Nitric acid in the range of 0.7% - 1.5% (v/v)

was selected as a medium with higher sensitivity and

stability for the determination of Pb and Cd. A 1% nitric

acid was used in this work.

3.2. Matrix Modification Effect

The modification effects of several chemicals, such as

NH4NO3, (NH4)2SO4, NH4H2PO4 and PdCl2 as well as

NH4H2PO4 + Mg(NO3)2, on the determinations of Pb and

Cd were investigated comparatively. The absorbance-

pyrolysis temperature curves are shown in Figure 1.

Using NH4H2PO4 as a modifier, the critical pyrolysis

temperature for the determination of Pb was raised from

300˚C to 900˚C with a higher sensitivity and lower back-

ground absorption because of the formations of more

stable lead phosphate and easy volatile ammonium chlo-

ride in the presence of NH4H2PO4. (NH4)H2PO4 was used

as a matrix modifier for Cd determination [8]. In our

work, use of NH4H2PO4 plus Mg(NO3)2 as a matrix

Figure 1. The absorbance- pyrolysis temperature curves. a.

Pb + NH4H2PO4, b. Pb, c. Cd + NH4 H2PO4 +Mg(NO3)2, d.

Cd. Data point is the average value of three measurements

with < 2% of RSD.

H. W. SUN ET AL.

220

modifier made the critical pyrolysis temperature of Cd

raised from 400˚C to 800˚C, and obtained higher sensi-

tivity than NH4H2PO4 alone. Thus, the optimum pyroly-

sis temperature of 800˚C was chosen for the determina-

tion of Pb and Cd in the presence of the modifiers.

The effects of modifier amounts on the sensitivity

were investigated. The sensitivity increased obviously

with the increase of the modifier concentration from 0 to

200 μg. The result showed that the best sensitivities

were achieved using 100 μg NH4H2PO4 and 120 μg

NH4H2PO4 + 5 μg Mg(NO3)2 for the determination of Pb

and Cd , respectively. Plateaus of the atomization tem-

perature were observed over 1700˚C - 2000˚C using

NH4H2PO4 and NH4H2PO4 + Mg(NO3)2 modifiers, re-

spectively. Therefore, the optimal atomization tempera-

tures were selected as 1700˚C for Pb and 1800˚C for Cd.

In addition, the “maximum power” and gas stop modes

were used for the determination of Pb and Cd in order to

obtain high sensitivity.

3.3. Linearity, Detection Limit and Precision

The calibration graph was linear up to 70 µg·L–1 for Pb

and up to 4 µg·L–1 for Cd. Linearity relation between

absorbance (A) and concentrations (C) can be described

by a regression equation: for Pb, A = 454.802C – 2.600,

and for Cd, A = 37.068C – 0.123, with a correlation co-

efficient of > 0.999. The detection limit calculated ac-

cording to the IUPAC rules on the basis of 3σ criterion

for 11 replicate measurements of the blank signal was

0.21 µg·L–1 for Pb and 0.005 µg·L–1 for Cd (4.2 pg Pb

and 0.1 pg Cd for sampling 20 µL solutions). For 1 g

sample and 50 mL final solution, the method LOD was

10.5 µg·kg–1 for Pb and 0.25 µg·kg–1 for Cd. The relative

standard deviation (RSD, n = 7) was 1.54% for Pb at 40

ng·mL–1 level and 2.4% for Cd at 0.4 ng·mL–1 level. It is

indicated that the proposed method has good linearity,

high sensitivity and precision, and can permit the accu-

rate detection of trace Pb and Cd in herbs and their de-

coctions.

3.4. Determination of Pb and Cd in Herbs and

Their Decoctions

A water decoction is the one used for people to treat dis-

ease, and the content of Pb and Cd in the water decoction

was the actual contribution to the toxicity. The prepara-

tion of water decoctions and determination of total Pb/Cd

in herbs and total water-soluble Pb/Cd in water decoc-

tions were carried out according to the method described

in the procedure. The results are listed in Table 2.

The contents of Pb in the five herbs were higher than

for Cd in the same herbs. Though the content of total Pb

in Common anemarrhena rhizo me was the highest in the

five herbs, but the content of water-soluble Pb was the

lowest. In contrast, the contents of total Pb in Liquorice

was the lowest, but the contents of water-soluble Pb were

near to that in Common anemarrhena rhizome. It was

indicated that the dose of trace element taken in human

body could not be evaluated with total content of the

element in herb.

The practical feasibility of the proposed system was

tested on the five kinds of Chinese herbal medicines.

Since standard reference materials with certified Pb and

Cd values were not available, the selectivity and accu-

racy of the method were tested by a standard spiking

method. The recovery test was carried out in the same

way for spiked samples in the range of 0.15 - 10 µg·g–1

for Pb, and 0.01 - 0.2 µg·g–1 for Cd (each added content

nears real content in the sample). The recoveries of ana-

lyte from Common anemarrhena rhizome, Liquorice,

Dioscorea nippon ica makino, Rad ix sa lviae miltio rrh izae

and Fructus ligustri lucidi were 98.6, 99.3, 96.9, 102 and

98.4% for total Pb, and 97.3, 98.7, 95.8, 97.7 and 98.9%

for total water-soluble Pb, and 97.9, 100, 99.3, 98.8 and

96.9% for total Cd, and 98.9, 97.3, 98.6, 101 and 98.5%

for total water-soluble Cd, respectively. The data showed

that recovery was in the range of 95.8% - 102% for Pb

and 96.9% - 101% for Cd. The relative standard devia-

tions (R.S.D., n = 3) were in the range 1.7% - 3.4%,

demonstrating the general reproducibility of this method

Table 2. Determination of total Pb/Cd and total water-soluble Pb/Cd in herbs and their decoctions ( n = 3).

Sample Total Pb

(µg g–1)

Total Cd

(µg g–1)

Total water-

soluble Pb

(µg·g–1)

Total water-

soluble Cd

(µg·g–1)

Dissolving ratio

Pb Cd

(%) (%)

Common anemarrhena

rhizome 9.29 0.19 0.17 0.01 1.83 5.26

Liquorice 1.54 0.15 0.18 0.01 11.7 6.04

Dioscorea nipponica makino 4.77 0.14 0.38 0.03 7.97 21.4

Radix salviae miltiorrhizae, 4.94 0.19 0.25 0.02 5.06 10.5

Fructus ligustri lucidi 5.11 0.21 1.21 0.02 23.7 9.52

H. W. SUN ET AL.

221

3.5. Dissolving Capability

Based on above result, water-dissolving capability of the

analytes was examined. Table 2 showed the dissoluble

ratios (total water-soluble Pb/Cd contents to total Pb/Cd

content). The dissoluble ratio was related to the kind of

herb and element. For Fructus ligustri lucidi, the dis-

solving ratio of Pb was the highest. Although the content

of Pb in Common anemarrhena rhizome was the highest,

the dissolving ratio was the lowest. The content of Cd in

Dioscorea nipponica makino was the lowest, and the

dissolving ratio also was the highest. For Common ane-

marrhena rhizome the dissolving ratio of Cd was the

lowest. Otherwise, except for Liquorice and Fructus li-

gustri lucidi, the dissolving ratios of Cd were higher than

for Pb in the same herbs. The dissolving capability of Pb

and Cd in each of the five herbs has their own distributed

regularity. The kind of herb has a strong relationship to

the dissolving ratios.

3.6. Distribution of Pb and Cd in Water

Decoction at Stomach and Intestine

Acidities

A decoction is one of the well known herbal preparations

that are used for healing many diseases and ailments.

The stomach and intestine are the main absorption or-

gans in the human body. The n-octanol-soluble Pb and

Cd have stronger lipophilic and biological activity. Since

the structure of n-octanol is similar to aqua-carbon com-

pound and ipide’s structure, so the distribution of Pb and

Cd in water decoction at stomach and intestinal acidity in

the presence of n-octanol is represented by their distribu-

tion in the stomach and intestine.

The content ratio ow (kowo w

kcc, where o is n-

octanol-soluble content; w is water-soluble content)

was employed to evaluate the lipophilic and biological

behaviors of organic compounds [17]. In this study, the

concentrations of water-soluble and n-octanol-soluble Pb

and Cd under gastric acidity (pH = 1.3) and intestinal

acidity (pH = 7.6) conditions were investigated. The re-

sults are listed in Table 3.

For the studied herbs, ow for Pb and Cd under intes-

tinal acidity was higher than that under gastric acidity, it

showed that analyte would be removed from water-

soluble to n-octanol-soluble Pb with raising pH of water

decoction. Otherwise, ow values for the five herbs also

had some differences. It is indicated that the content of

water-soluble and n-octanol-soluble analyte and its ratio

were related with the kind of herbal medicine and the

acidity of the decoction.

When , the quantum of n-octanol-soluble Pb

and Cd taken in gastric and intestinal part would be

higher than that of water-soluble Pb and Cd. When kow <

1, it was reverse. Therefore, when pharmacology actions

of herbal medicines were studied, medicine effects of

water decoction would not be evaluated only with total

content of Pb or Cd in water decoction. The component

of medicines, acted target, acidity of target and compati-

bility of medicines should be taken into account when

studying toxicology of herbal medicine.

k

4. Conclusions

It was indicated that the dose of trace element taken in

human body could not be evaluated with total content of

Table 3. Distribution of water-soluble and n-octanol-soluble Pb and Cd at gastric and intestinal acidity (n = 3)

Stomach acidity (pH 1.3) Intestine acidity (pH 7.6)

Sample Element

Water-

Soluble

n-octanol-

soluble ow

k Water-

Soluble

n-octanol-

soluble ow

Pb 0.098 0.083 0.85 0.059 0.123 2.10

Common anemarrhena

rhizome Cd 0.008 0.004 0.50 0.004 0.009 1.78

Pb 0.073 0.097 1.33 0.060 0.111 1.82

Liquoric

Cd 0.007 0.003 0.42 0.007 0.003 0.45

Pb 0.360 0.017 0.05 0.246 0.131 0.53

Dioscorea nipponica makino

Cd 0.010 0.004 0.40 0.008 0.007 0.82

Pb 0.226 0.022 0.10 0.215 0.034 0.16

Radix salviae miltiorrhizae,

Cd 0.009 0.004 0.45 0.007 0.007 1.00

Pb 0.878 0.333 0.40 0.806 0.406 0.50

Fructus ligustri lucidi

Cd 0.015 0.009 0.62 0.017 0.011 0.65

H. W. SUN ET AL.

222

the element in herb. A n-octanol system can be used to

study the distribution of Pb and Cd in water decoction in

our stomach and intestine. By using NH4H2PO4 as a

modifier for Pb and NH4H2PO4 plus Mg(NO3)2 as a ma-

trix modifier for Cd, the critical pyrolysis temperature

could be raised to 900˚C and 800˚C, respectively, assur-

ing high analytical sensitivity and lower background ab-

sorption. The detection limits of the proposed method for

Pb and Cd were lower than that of FAAS [3,4], GFAAS

[5,6,8,10] and HGAFS methods[11]. The proposed me-

thod is a reliable and effective for the determination of

Pb and Cd in Chinese herbal medicines and their de-

coctions. The distribution of Pb and Cd in water decoc-

tion at stomach and intestine acidities was developed in

the first time. The proposed method can help us make a

further study on the chemical and biological behaviors,

regularity of elements movement and toxicity of Pb and

Cd in Chinese herbal medicines, and gives some advice

on Chinese traditional herbs’ plant and use.

5. Acknowledgements

We express thanks to the Natural Science Foundation of

Hebei province of China for much support to the studied

subject (B2008000583).

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