Rapid Analysis of Aristolochic Acid Analogues in Traditional Chinese Patent Medicine by LC-MS/MS

Abstract

Aristolochic acids have been demonstrated to have renal toxicity, cause carcinogenesis, and may cause gene mutations. A series of risk control measurements have been adopted worldwide since 1990s. Some varieties of traditional Chinese medicine with high content of aristolochic acids have been banned in China. However, some species containing aristolochic acids in microscale are still in use. In recent years, with the continuous awareness of drug safety, the aristolochic acid analogues were generally considered to be of potential safety risks. Among these constituents, aristolochic acid I is still the one with most studies. Therefore, in addition to aristolochic acid I, it is necessary to establish an accurate and rapid method to determine other aristolochic acid analogues. LC-MS/MS methods based on multireaction monitoring mode was established to simultaneously determine 9 aristolochic acid analogues including 5 aristolochic acids and 4 aristolactams for the first time. Furthermore, the method was applied for Long dan Xie gan Pill, a traditional complex compound preparation with a long history for treatment of diseases including hepatochlic hygropyrexia, dizziness, tinnitus, and deafness. It has attracted widespread attention because of the aristolochic acid nephropathy. The crude drug Caulis Aristolochiae manshuriensis (Guanmutong) collected in the prescription was replaced by Akebiae Caulis (Mutong), and the established method helps to understand the product safety on market. As a result, aristolochic acid I, aristolochic acid Iva, and aristolactam I were detected and determined in one batch of Long dan Xie gan Pill among 25 batches of samples. It provided practical approach to demonstrate trace aristolochic acids and aristolactams. It was beneficial to control the safety of related traditional Chinese medicine products.

Full text

Research Article
Rapid Analysis of Aristolochic Acid Analogues in Traditional
Chinese Patent Medicine by LC-MS/MS
Jing Liu , Yang Liu, Yingxue Wu, Zhong Dai , and Shuangcheng Ma
National Institutes for Food and Drug Control, Beijing 100050, China
Correspondence should be addressed to Zhong Dai; daizhong@nifdc.org.cn and Shuangcheng Ma; masc@nifdc.org.cn
Received 13 August 2020; Revised 18 September 2020; Accepted 6 November 2020; Published 19 November 2020
Academic Editor: Mohamed Abdel-Rehim
Copyright ©2020 Jing Liu et al. is is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Aristolochic acids have been demonstrated to have renal toxicity, cause carcinogenesis, and may cause gene mutations. A series of
risk control measurements have been adopted worldwide since 1990s. Some varieties of traditional Chinese medicine with high
content of aristolochic acids have been banned in China. However, some species containing aristolochic acids in microscale are
still in use. In recent years, with the continuous awareness of drug safety, the aristolochic acid analogues were generally considered
to be of potential safety risks. Among these constituents, aristolochic acid I is still the one with most studies. erefore, in addition
to aristolochic acid I, it is necessary to establish an accurate and rapid method to determine other aristolochic acid analogues. LC-
MS/MS methods based on multireaction monitoring mode was established to simultaneously determine 9 aristolochic acid
analogues including 5 aristolochic acids and 4 aristolactams for the first time. Furthermore, the method was applied for Long dan
Xie gan Pill, a traditional complex compound preparation with a long history for treatment of diseases including hepatochlic
hygropyrexia, dizziness, tinnitus, and deafness. It has attracted widespread attention because of the aristolochic acid nephropathy.
e crude drug Caulis Aristolochiae manshuriensis (Guanmutong) collected in the prescription was replaced by Akebiae Caulis
(Mutong), and the established method helps to understand the product safety on market. As a result, aristolochic acid I,
aristolochic acid Iva, and aristolactam I were detected and determined in one batch of Long dan Xie gan Pill among 25 batches of
samples. It provided practical approach to demonstrate trace aristolochic acids and aristolactams. It was beneficial to control the
safety of related traditional Chinese medicine products.
1. Introduction
Aristolochic acid (AA) analogues are a kind of compounds
with nitrophenanthrene core structure, including aristolo-
chic acids and aristolactams (ALs). ese compounds
mainly exist in the genus of Aristolochia and Asarum from
the family of Aristolochiaceae plants. It has been proved that
some aristolochic acids have renal toxicity, cause carcino-
genesis, and may cause gene mutations [1–6]. us, a series
of risk control measurements have been adopted worldwide
since 1990s [7–9]. ree varieties of Caulis Aristolochiae
manshuriensis, Radix Aristolochia fangchi, and Radix
Aristolochiae with high content of aristolochic acids have
been banned in China since 2003. However, some Chinese
herbal medicines and preparations with trace amount of
aristolochic acids are still in use. Until now, most of the toxic
studies are focused on AA I. Due to the serious adverse effect
of AAs and the insufficient research on other aristolochic
acid analogues except for AA I, it is essential to develop a
simple and fast method to identify and quantify the AA
analogues in commonly-used products. Since the structures
of these AA analogues are similar and some are isomers with
different substitution positions of hydroxyl and/or methoxy
groups, it is challenging to qualify and quantify the AA
analogues at the same time.
At present, different methods have been reported for de-
termination of AAs, including thin-layer chromatography
(TLC), high performance liquid chromatography (HPLC),
liquid chromatography-mass spectrometry (LC-MS), enzyme-
linked immunosorbent assay (ELISA), and capillary electro-
phoresis (CE) [9–11]. e HPLC and LC-MS methods are also
widely used in characterizing the AA analogues [12–18]. HPLC
Hindawi
Journal of Analytical Methods in Chemistry
Volume 2020, Article ID 8823596, 7 pages
https://doi.org/10.1155/2020/8823596

is more suitable for those with higher content of AA analogues.
For traditional Chinese patent medicines with trace amount of
AA analogues or with component interference, LC-MS shows
strong advantages of high specificity and high sensitivity. LC-
MS has been applied for qualification by MS and MS/MS
spectra comparison. Meanwhile, it is also used for quantification
mainly based on the extracted ion chromatogram (EIC) mode.
Also, the reported multireaction monitoring (MRM) mode was
mainly focused on AA I and/or AA II. In this study, we report
that the new LC-MS/MS method is capable of characterizing 9
AA analogues (Figure 1) including aristolochic acid I, aristo-
lochic acid II, aristolochic acid IIIa, aristolochic acid IVa, 7-
hydroxy aristolochic acid I, aristolactam I, aristolactam AIIIa,
aristolactam BII, and aristolactam FI for the first time.
Moreover, the LC-MS/MS method was further applied to
Long dan Xie gan Pill, a commonly-used traditional Chinese
patent medicine, with a long history for treatment of diseases
including hepatochlic hygropyrexia, dizziness, tinnitus, and
deafness [19, 20]. Long dan Xie gan Pill has attracted widespread
attention since it caused aristolochic acid nephropathy (AAN)
by Caulis Aristolochiae manshuriensis (Guanmutong) which
existed in the prescription before 2003 [21]. en, toxic
Guanmutong was replaced by Akebiae Caulis (Mutong)
without AAs inside. In order to understand whether there is still
safety risk of raw material adulteration and further investigate
the product safety, 25 batches of Long dan Xie gan Pills from 8
enterprises were analyzed. e results indicated that the
established method could efficiently analyze the aristolochic
acid analogues for qualitative and quantitative purposes. Also, it
could provide a practical approach for related species, especially
traditional Chinese patent medicines containing trace content
of aristolochic acid analogue constituents. e LC-MS/MS
method reported in this paper was valuable for the safety control
of related traditional Chinese medicine products.
2. Materials and Methods
2.1. Chemicals and Reagents. AA I (99.1%, Lot no.
110746–201912) was from National Institutes for Food and
Drug Control, Beijing, China. AA II (HPLC purity≥98%, Lot
no. P13J10F90613), AA IIIa (HPLC purity≥98%, Lot no.
P20N8F48641), AA IVa (HPLC purity≥98%, Lot no.
Z03J10X89866), 7-hydroxy AAI (HPLC purity≥95%, Lot No.
Z13N9S74959), AL I (HPLC purity≥98%, Lot no.
P27N10S104067), AL AIIIa (HPLC purity≥98%, Lot no.
T09M11Z112630), AL BII (HPLC purity≥97%, Lot no.
X09M11_112631), and AL FI (HPLC purity≥98%, Lot no.
X09M11L112632) were from Shanghai Yuanye Bio-Technol-
ogy Co., Ltd. Methanol (analytical reagent) was from National
Drug Chemical Reagents Co., Ltd. Acetonitrile (chromato-
graphic pure), formic acid (mass spectrometry reagent), and
ammonium acetate (mass spectrometry reagent) were from
ermo Fisher Scientific. Water was of ultrahigh purity.
2.2. Materials. 25 batches of Long dan Xie gan Pill
(LDXGW) samples were from 8 manufacturers (A-H, batch
no. A01-A04, B01-B03, C01-C05, D01-D05, E01-E05, F01,
G01, and H01).
2.3. Instrumentation. An Agilent 1260–6410B triple quad-
rupole LC-MS system (Agilent Technologies, Inc., Santa
Clara, CA, USA) equipped with an electrospray ionization
device was used for sample analysis. METTLER XS105
electronic analytical balance (Mettler-Toledo, Zurich,
Switzerland), Milli-Q water purification system (Millipore,
Burlington, USA), and KQ-300DA numerical control ul-
trasound cleaning instrument (Kun Shan Ultrasonic In-
struments Co., Ltd., Kunshan, China) were used.
2.4. Preparation of Standard Solutions. Standard stock so-
lutions of aristolochic acid I, aristolochic acid II, aristolochic
acid IIIa, aristolochic acid IVa, 7-hydroxy aristolochic acid I,
aristolactam I, aristolactam AIIIa, aristolactam BII, and
aristolactam FI were prepared by dissolving suitable
amounts of reference substance in methanol to make the
concentration at 5 μg·mL
−1
, respectively.
2.5. Preparation of Sample Solutions. For Long dan Xie gan
Pill (6 g per small bag), 5 bags were mixed and pulverized to
powder. en, 2 g was weighed accurately and put into a
50 mL plug conical bottle. Twenty-five mL methanol was
added precisely and weighed, respectively. After extracting
by ultrasonic extraction (power: 300 W; frequency: 40 kHz)
for 30 min, the extract was cooled down and then made up
for lost weight by adding methanol. e continuous filtrate
was taken and then filtered by 0.22 μm microporous filter
membrane.
2.6. HPLC Chromatographic Condition. Column: Agilent
SB-C18 (2.1 ×50 mm, 1.8 μm); mobile phase: gradient elu-
tion with acetonitrile (A)-0.1% formic acid solution (con-
taining 5 mmol·L
−1
ammonium acetate) (B) (0–15 min, 31%
A; 15–18 min, 31% A-60%A; 18–20 min, 60%A; 20–22 min,
60%A-31%A); flow rate: 0.3 mL·min
−1
; column temperature:
30°C; injection volume: 2 µL.
2.7. MS Condition. e triple quadrupole MS equipped with
a positive electrospray ionization source was used in the
MRM mode [22]. e equipment was set with a drying gas
flow, nebulizer pressure, gas temperature, and spray voltage
of 9 L·min, 30 psi, 350°C, and 4000 V, respectively.
e MRM conditions were individually optimized for each
of the nine aristolochic acid analogues reference standards (AA
I, AA II, AA IIIa, AA IVa, 7-OH AAI, AL I, AL AIIIa, AL BII,
and AL FI) on account of their different structures [22]. e MS
conditions for MRM are summarized in Table 1, and the typical
MRM chromatogram is shown in Figure 2.
3. Result
e established LC-MS/MS method for 9 aristolochic acid
analogues was applied to Long dan Xie gan Pills, and two
aristolochic acids (AA I and AA IVa) and one aristolactam
(AL I) were determined (Figure 3).
3.1. Linearity. Working standard solutions containing AA
IVa, AL I, and AA I were prepared by diluting the stock
mixed solution with methanol to a series of proper
2Journal of Analytical Methods in Chemistry

concentrations. en, they were injected and analyzed. e
results of regression equations, linearity, determination
coefficient, and limits of detection and quantification of the
method are presented in Table 2. All analytes presented a
determination coefficient (R
2
) of the 0.999 which allows the
method to be considered linear.
3.2. Limit of Detection and Limit of Quantification.
Precisely dilute the stock mixed solution with methanol
quantitatively and stepwise if necessary. e diluted solu-
tions were separately injected and analyzed. e limits of
detection (LOD) and quantification (LOQ) (Table 3) were
defined as the concentrations that could be detected and
yield signal-to-noise (S/N) ratios of 3 : 1 and 10 : 1, respec-
tively, according to guidelines for validation of analytical
methods for pharmaceutical quality standards [23].
3.3. Instrument Precision. e same sample solution (A04)
was injected for six consecutive times and analyzed. e
RSDs of peak areas for AA IVa, AL I, and AA I were 5.13%,
4.62%, and 1.80%, respectively. It indicated the precision of
the instrument was in accordance with requirement in
guidelines for validation of analytical methods for phar-
maceutical quality standards [23].
O
O
O
COOH COOH
NO2NO2NO2NO2
COOH COOH
NO2
COOH
O
O
O
O
HO HO
O
O
OH
O
O
AAI AAII AAIIIa AAIVa 7-OHAAI
AL AIIIa
O
AL BIIALI AL FI
O
O
OH3CO
H3CO
H3CO
H3CO
HO
HO
HO
NH
NH
NH
NH
O
OCH3
OCH3
OCH3
OCH3
Figure 1: Chemical structures of aristolochic acid analogues.
Table 1: MS parameters for 9 AA analogues reference standards.
No. Compound Retention time (min) Precursor ion (m/z) Production (m/z) Fragmentor (V) CE (eV)
1 AL AIIIa 1.37 282.0 265.0∗80 10
250.0 80 20
2 AA IIIa 2.32 345.0 282.0∗60 8
284.0 60 5
3 7-OH AAI 2.94 375.0 314.0∗65 8
340 65 10
4 AA IVa 3.12 375.0 312.0∗65 8
314.0 65 8
5 AL FI 3.93 266.0 251.0∗125 25
195 125 30
6 AA II 9.21 329.0 268.0∗70 8
294.0 70 5
7 AL BII 9.85 280.0 264.0∗100 30
236.0 100 30
8 AL I 10.43 294.0 279.0∗120 30
251.0 120 40
9 AA I 13.08 359.0 298.0∗65 5
296.0 65 5
∗
e quantitative ions.
Journal of Analytical Methods in Chemistry 3

3.4. Repeatability. e same batch of sample (A04) was
taken and prepared for six independent sample solutions.
en, they were analyzed according to conditions under 2.6
and 2.7. e average contents of AA IVa, AL I, and AA I
were 0.27, 0.27, 2.92 mg ·g
−1
, respectively, and the RSDs were
3.75%, 6.16%, and 5.38%, respectively. It indicated method
×104
6
4
1.368
2
0
(a)
×103
1
2.5
2
1.5
1
0.5
0
2.318
(b)
1
×103
2.5
3
2
1.5
1
0.5
0
2.943
(c)
1
×103
2.5
2
1.5
1
0.5
0
3.116
(d)
1
×104
2.5
2
1.5
1
0.5
0
(e)
×103
2.5
2
1.5
1
0.5
0
(f)
1
×103
5
4
3
2
1
0
(g)
1
×104
2.5
2
1.5
1
0.5
0
10.428
(h)
1
Counts vs acquisition time (min)
0123456789101112131415
×103
2.5
2
1.5
1
0.5
0
∗13.080
(i)
Figure 2: Typical MRM chromatograms of 9 aristolochic acid analogues reference standards. (a) AL AIIIa, (b) AA IIIa, (c) 7-OH AAI,
(d) AA IVa, (e) AL FI, (f ) AA II, (g) AL BII, (h) AL I, and (i) AA I.
R
Counts vs. acquisition time (min)
1234567891011121314151617181920
×103
×103
×103
7.5
5
2.5
0
5
2.5
0
5
0
1
1
1
∗3.126
∗10.945
∗12.835
(a)
S
Counts vs. acquisition time (min)
1234567891011121314151617181920
×103
2
1
0
×103
1
0
×104
0.5
11
1
1
0
∗3.178
13.042
10.980
(b)
Figure 3: MRM chromatograms of reference standards and LDXGW sample (R: reference standards; S: sample A04).
4Journal of Analytical Methods in Chemistry

repeatability was in accordance with requirement in
guidelines for validation of analytical methods for phar-
maceutical quality standards [23].
3.5. Stability. e same sample solution (A04) was injected
at 0, 4, 8, 12, and 20 h at room temperature. e RSDs of
peak areas for AA IVa, AL I, and AA I were 5.13%, 6.34%,
and 4.74%, respectively. It indicated the sample solution was
stable within 20 h.
3.6. Recovery. e recovery experiment was performed by
adding a known amount of individual reference standards
into a certain amount of sample (A04). Six separate samples of
1 g (contents of AA IVa, AL I, and AA I were 0.27, 0.27,
2.92 mg·g
−1
, respectively) were weighed accurately, and 1 mL of
mixed reference standard solution (concentrations of AA IVa,
AL I, and AA I were 0.5292, 0. 6811, 5.3068μg·mL
−1
, re-
spectively) was added then prepared samples according to 2.5.
e results (Table 4) showed that the average recoveries ranged
from 117.66% to 124.22% with RSDs in the range of 5.25%–
6.04%, which indicated that the method was basically accurate.
3.7. Sample Analysis. Twenty-five batches of samples were
prepared and analyzed according to 2.5, 2.6, and 2.7. e
results displayed that sample A04 contained AA IVa, AL I,
and AA I at the concentrations of 0.28, 0.25, and 2.67 μg·g
−1
,
respectively. No aristolochic acids and aristolactams were
detected in the other 24 samples.
4. Discussion
4.1. Optimization of HPLC Chromatographic Conditions.
In our study, different mobile phase systems including
acetonitrile-water, acetonitrile-0.1% formic acid, and
acetonitrile-0.1% formic acid (containing 5mM ammo-
nium acetate) were investigated. As a result, the ioni-
zation intensities of aristolochic acids were best
with ammonium acetate added in the mobile phase. us,
the mobile phase including ammonium acetate was
adopted.
4.2. Optimization of MS Conditions. During optimization of
MS conditions for nine aristolochic acid analogues, it was
shown that the precursor ions for the five aristolochic acids
were all in the form of [M + NH
4
]
+
, while those for the four
aristolactams were all in the form of [M + H]
+
. is could be
caused by the structural difference between aristolochic
acids and aristolactams, and the main difference was that the
existence form of nitrogen was nitro group and secondary
amino group, respectively.
Further analysis indicated that both the product ions for
quantitative and qualitative purpose of the five aristolochic
acids were the fragmentation ions corresponding to [M + H-
NO
2
]
+
, [M + H-CO
2
]
+,
and [M + H-H
2
O]
+
. Although AA
IVa and 7-OH AA I were isomers, their product ions were
different due to the different substitution of hydroxyl and
methoxyl groups. Also, the fragment ion at m/z 314 was to
quantity for 7-OH AA I, but it was used to qualify for AA
IVa. It should be mentioned that they could not be extracted
at the same injection.
4.3. Result Discussion. Since there was no aristolochic acids
contained in Akebiae Caulis (Mutong) in Long dan Xie gan
Pill prescription, aristolochic acid I should not be detected.
e determination results indicated that there was no
substitution problem. It was possible that Akebiae Caulis
(Mutong) was mixed with Caulis Aristolochiae manshur-
iensis (Guanmutong). erefore, we should strictly control
the quality of raw medicinal materials. In view of the harm of
aristolochic acid I to human, the method has been applied
for supplementary inspection method, and it provided
sound scientific basis for further risk control measures and
safeguard for drug safety.
4.4. Determination of Aristolochic Acid Analogues. Until
now, there are more than 80 AAs and ALs found from the
Aristolochiaceae family [24], and it would be much better if
all these constituents could be determined. However, there
are many limitations to accomplish this work. For quanti-
tative analysis, reference substance is necessary to ensure the
result accuracy. So far, there are only a few AAs and ALs
reference substances. Furthermore, more than 80 AAs and
ALs were mainly from the related crude drugs. eir con-
tents were quite different, and many of them may exist in
trace amounts. For traditional Chinese patent medicines that
may contain AAs and ALs, the related crude drugs con-
taining AAs and ALs were in low proportion of prescrip-
tions. Based on our previous study and the literature
research, the AAs and ALs with comparatively higher
Table 2: Regression equations, linear ranges, and coefficients for AA IVa, AL I, and AA I.
Components Regression equations Linear ranges (pg) R
2
AA IVa y�2.3306x−1.2291 9.702∼116.424 0.9992
AL I y�9.0052x−21.389 13.524∼162.288 0.9996
AA I y�4.6873 – 64.752 96.6225∼1159.47 0.9992
Table 3: LODs and LOQs for 9 aristolochic acid analogues.
Components AL AIIIa AA IIIa 7-OH AAI AA IVa AL FI AA II AL BII AL I AA I
LODs (pg) 0.25 7.80 1.44 4.62 1.62 8.75 4.95 5.52 8.12
LOQs (pg) 0.74 23.40 4.32 13.85 4.85 26.25 14.85 16.56 24.38
Journal of Analytical Methods in Chemistry 5

contents in crude drugs were studied in this manuscript.
Considering the accuracy of detection results and re-
search-related species, the LC-MS/MS determination
method based on MRM mode for AL AIIIa, AA IIIa, 7-OH
AAI, AA IVa, AL FI, AA II, AL BII, AL I, and AA I was
established.
5. Conclusions
In this study, an efficient LC-MS/MS method based on MRM
for 9 aristolochic acid analogues was established. It was also
successfully applied for the commonly-used traditional
Chinese patent medicine, Long dan Xie gan Pill. Since MRM
has the outstanding advantages of strong specificity, high
sensitivity, high accuracy, good reproducibility, and high
throughput automation, the established method was dem-
onstrated to be powerful for determination of the trace
aristolochic acid analogues in complex traditional Chinese
medicine. It also could provide scientific basis for the follow-
up safety risk control measures.
Data Availability
e electronic file of data used to support the findings of this
study are available from the corresponding author upon
request.
Conflicts of Interest
e authors declare that there are no conflicts of Interest
regarding the publication of this paper.
Authors’ Contributions
Jing Liu and Yang Liu contributed equally to this
manuscript.
Acknowledgments
e investigation was financially supported by China Food
and Drug Administration consignment inspection project.
References
[1] P. Balachandran, F. Wei, R.-C. Lin, I. A. Khan, and
D. S. Pasco, “Structure activity relationships of aristolochic
acid analogues: toxicity in cultured renal epithelial cells,”
Kidney International, vol. 67, no. 5, pp. 1797–1805, 2005.
[2] H.-B. Song, J.-T. Ren, L. Yang, X.-X. Guo, and X.-X. Du,
“Advance in studies on toxicity of aristolochic acid and
analysis on risk factors,” China Journal of Chinese Materia
Medica, vol. 39, no. 12, pp. 2246–2250, 2014.
[3] J. Michl, G. C. Kite, S. Wanke et al., “LC-MS- and1H NMR-
based metabolomic analysis and in vitro toxicological as-
sessment of 43AristolochiaSpecies,” Journal of Natural
Products, vol. 79, no. 1, pp. 30–37, 2016.
[4] Y. Gao, X.-H. Xiao, X.-X. Zhu, A.-H. Liang, and B.-L. Zhang,
“Study and opinion on toxicity of aristolochic acid,” China
Journal of Chinese Materia Medica, vol. 42, no. 21,
pp. 4049–4053, 2017.
[5] Y.-M. Li, J. Li, and F. Yu, “Current situation and research
progress on the nephrotoxicity if aristolochic acid com-
pounds,” Journal of Pharmaceutical Research, vol. 36, no. 9,
pp. 534–536, 2017.
[6] A. W. T. Ng, S. L. Poon, M. N. Huang et al., “Aristolochic
acids and their derivatives are widely implicated in liver
cancers in Taiwan and throughout Asia,” Science Translational
Medicine, vol. 9, no. 412, pp. eaan6446–12, 2017.
[7] Z.-F. Bai, G. Xu, J.-B. Wang, and X.-H. Xiao, “Researcn
progress in and countermeasures against carcinogenicity of
aristolochic acids and their derivatives,” Progress in Phar-
maceutical Science, vol. 42, no. 3, pp. 164–169, 2018.
[8] J.-Z. Tian, A.-H. Liang, J. Liu, and B.-L. Zhang, “Risk control
of traditional Chinese medicines containing aristolochis acids
(AAs) based on influencing factors of content of AAs,” China
Journal of Chinese Materia Medica, vol. 42, no. 24,
pp. 4679–4686, 2017.
Table 4: Recovery results of AA IVa, AL I, and AA I in Long dan Xie gan Pill samples.
Components No. Sampling amount
(g)
Sample content
(μg)
Added amount
(μg)
Detected amount
(μg)
Recovery
(%)
Average recovery
(%)
AA IVa
1 1.1835 0.3195
0.5292
0.9575 120.55
124.22% (RSD
5.25%)
2 1.1586 0.3128 0.9311 116.84
3 1.1270 0.3050 0.9509 122.04
4 1.0938 0.2953 0.9452 122.81
5 1.1324 0.3057 1.0226 135.47
6 1.1981 0.3235 0.9988 127.62
AL I
1 1.1835 0.3195
0.6811
1.1064 115.52
120.85% (RSD
6.04%)
2 1.1586 0.3128 1.2322 134.98
3 1.1270 0.3050 1.0925 115.62
4 1.0938 0.2953 1.1221 121.38
5 1.1324 0.3057 1.1083 117.83
6 1.1981 0.3235 1.1391 119.75
AA I
1 1.1835 3.4558
5.3068
9.0727 105.84
117.66% (RSD
5.39%)
2 1.1586 3.3831 9.6685 118.44
3 1.1270 3.2987 9.7298 121.19
4 1.0938 3.1939 9.7422 123.39
5 1.1324 3.3066 9.7375 121.18
6 1.1981 3.4985 9.6487 115.89
6Journal of Analytical Methods in Chemistry

[9] J. Liu, R.-X. Guo, Z. Dai, and S.-C. Ma, “Research progress on
aristolochic acids,” World Science and Technlogy/Moderni-
zation of Traditional Chinese Medicine and Materia Medica,
vol. 21, no. 7, pp. 1280–1286, 2019.
[10] J. Liu, Z. Dai, X.-L. Cheng, F. Wei, and S.-C. Ma, “Research
progress on detection and analysis of aristolochic acids,” Drug
Evaluation Research, vol. 42, no. 8, pp. 1644–1650, 2019.
[11] D.-Y. Yao, L. Wang, H.-M. Ma, and B.-L. Zhang, “Research
and application of modern analytical technology in the de-
tection of aristolochic acids,” West China Journal of Phar-
maceutical Sciences, vol. 22, no. 5, pp. 542–544, 2007.
[12] L. Vaclavik, A. J. Krynitsky, and J. I. Rader, “Quantification of
aristolochic acids I and II in herbal dietary supplements by
ultra-high-performance liquid chromatography-multistage
fragmentation mass spectrometry,” Food Additives & Con-
taminants: Part A, vol. 31, no. 5, pp. 784–791, 2014.
[13] H.-M. Tian, X.-M. Cheng, C.-H. Wang, and Z.-T. Wang,
“Simultaneous determination of four constituents in Aris-
tolochia debilis by HPLC,” Chinese Traditional Patent Med-
icine, vol. 38, no. 3, pp. 560–565, 2016.
[14] D.-Q. Kong, H.-Y. Gao, X. Li, J.-C. Lu, and D. Yuan, “Rapid
identification of eight aristolochic acid analogues in five
Aristolochiaceae plants by ultra-high performance liquid
chromatography quadrupole/time-of-flight mass spectrome-
try,” Journal of Chinese Pharmaceutical Science, vol. 24, no. 6,
pp. 364–375, 2015.
[15] W.-W. Mao, W. Gao, Z.-T. Liang, P. Li, Z.-Z. Zhao, and
H.-J. Li, “Characterization and quantitation of aristolochic
acid analogues in different parts of Aristolochiae Fructus,
using UHPLC-Q/TOF-MS and UHPLC-QqQ-MS,” Chinese
Journal of Natural Medicines, vol. 15, no. 5, pp. 0392–0400,
2017.
[16] M. Araya, S. Garc´
ıa, and M. Gonz´alez-Teuber, “Rapid iden-
tification and simultaneous quantification of aristolochic
acids by HPLC-DAD and confirmations by MS in Aristo-
lochia chilensis using a limited biomass,” Journal of Analytical
Methods in Chemistry, vol. 2018, Article ID 5036542, 8 pages,
2018.
[17] C.-K. Chan, N. M. Pavlovi´c, and W. Chan, “Development of a
novel liquid chromatography-tandem mass spectrometric
method for aristolochic acids detection: application in food
and agricultural soil analyses,” Food Chemistry, vol. 289,
pp. 673–679, 2019.
[18] F.-Q. Ji, R.-R. Jin, C. Luo et al., “Fast determination of
aristolochic acid I (AAI) in traditional Chinese medicine soup
with magnetic solid-phase extraction by high performance
liquid chromatography,” Journal of Chromatography A,
vol. 1609, Article ID 460455, 2020.
[19] Chinese Pharmacopoeia Commission, Chinese
Pharmacopoeia, pp. 770-771, e Medicine Science and
Technology Press of China, Beijing, China, 2015.
[20] J. Liu, H. Liu, Z. Dai, and S. Ma, “Quality analysis of long dan
xie gan Pill by a combination of fingerprint and multicom-
ponent quantification with chemometrics analysis,” Journal of
Analytical Methods in Chemistry, vol. 2018, Article ID
4105092, 10 pages, 2018.
[21] H.-J. Chang, X.- Lian Qi, X.-L. Lin, and Y.-Q. Wang,
“Longdan Xiegan Wan- associated aristolochic acid ne-
phrosis,” Adverse Drug Reaction Journal, vol. 5, no. 1,
pp. 42-43, 2003.
[22] Y.-Y. Chen, S. Wu, and Q.-Y. Yang, “Development and
validation of LC-MS/MS for analyzing potential genotoxic
impurities in Pantoprazole starting materials,” Journal of
Analytical Methods in Chemistry, vol. 2020, pp.1–8, Article ID
6597363, 2020.
[23] Chinese Pharmacopoeia Commission, Chinese Pharmacopoeia,
pp. 374–377, e Medicine Science and Technology Press of
China, Beijing, China, 2015.
[24] J. Yu, C.-M. Ma, X. Wang et al., “Analysis of aristolochic acids,
aristololactams and their analogues using liquid chroma-
tography tandem mass spectrometry,” Chinese Journal of
Natural Medicines, vol. 14, no. 8, pp. 0626–0640.
Journal of Analytical Methods in Chemistry 7