Received: 22 January 2022 | Revised: 2 April 2022 | Accepted: 12 April 2022

DOI: 10.1111/1556-4029.15046

PAPER
Toxicology

Diagnosis keracunan sianida yang mematikan.

Analisis dengan Kromatografi Penukar Anion dengan
Deteksi Amperometri Berdenyut
1 1 2
Pietro Zuccarello PhD | Giulia Carnazza SCM | Cataldo Raffino MD
1
| Nunziata Barbera MD
1
Department “G.F. Ingrassia”, Laboratory
of Forensic Toxicology, University of Abstract
Catania, Catania, Italy Cyanide is a poison widely used in cases of suicide or homicide. Although various methods to
2
Legal Medicine Center of INAIL, Enna,
Italy identify and quantify this substance are reported in the literature, they are mainly vali-dated on
biological fluids (e.g., blood and urine). In the present study, the Anion-Exchange Liquid
Correspondence
Pietro Zuccarello, Department “G.F. Chromatography with Pulsed Amperometric Detection (IC-PAD) method was vali-dated on
Ingrassia”, Laboratory of Forensic
blood and, for the first time, on gastric content, and organs (brain, lung, and liver). For each
Toxicology, University of Catania, Catania,
Italy. matrix, linearity, accuracy, precision, limit of detection (LOD), lower limit of quan-tification
Email: [email protected]
(LLOQ), matrix interferences, and carryover were assessed. The samples were extracted by
Funding information steam distillation in acid environment for the following analysis by IC-PAD. Furthermore,
University of Catania
cyanide values found in two real poisoning cases are reported. For each investigated matrix, the
analytical method satisfied all acceptance criteria for validation: it showed a good precision and
accuracy, selectivity, and sensitivity with no carryover and matrix interference. The extraction by
steam distillation in acid environment REDUCED the interference of the matrices and
ALLOWED to perform the analysis with good preci-sion and accuracy. In case #1, analysis
showed a blood cyanide concentration of 0.99 μg/ml. In case #2, cyanide concentrations were
1.3 μg/g in brain, 0.8 μg/g in lung, 1.6 μg/g in liver, and 1.2 μg/g in gastric content. The cyanide
concentrations found in the two reported cases have been suitable to cause death by poisoning.

KEYWORDS
blood, cyanide, forensic toxicology, gastric content, IC-PAD, organs, validation

Highlights
• Cyanide is currently used for homicide and suicide and diagnosis of lethal poisoning could
sometimes be complex.
• Method validation by IC-PAD on blood, gastric content, and organs (brain, lung, and liver)
was described.
• The analytical method satisfied all acceptance criteria for validation.
• Cyanide values found in two real poisoning cases are reported.

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in
any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2022 The Authors. Journal of Forensic Sciences published by Wiley Periodicals LLC on behalf of American Academy of Forensic Sciences.

J Forensic Sci. 2022;67:1617–1623. wileyonlinelibrary.com/journal/jfo | 1617

1618 | ZUCCARELLO et al.

1 | PERKENALAN 2.2 | Sample storage and extraction

Sianida (CN-) adalah garam hidrogen sianida (HCN). Karena toksisitas ex- Segera setelah otopsi, alikuot 1 ml darah atau 1 g kandungan otak, paru-paru,
hati, dan lambung dihomogenisasi dengan 5 ml NaOH 0,1 M dan disimpan pada
treme, sianida adalah salah satu racun yang paling ampuh; LD 50-nya kira-
suhu -20 ° C sampai analisis. Sampel diencerkan hingga 30 ml dengan air
kira 1-2 mg/kg berat badan [1]. Kalium sianida (KCN) dan natrium sianida
(NaCN) adalah bentuk sianida yang paling umum dicerna. Setelah ion ultramurni. Setiap sampel ditambahkan dengan 30 mg timbal sulfat yang

sianida mencapai lingkungan lambung yang sangat asam, CN-diubah digunakan untuk mengurangi konsentrasi sulfida. Setiap sampel dipindahkan ke

menjadi HCN, yang berdifusi secara pasif melintasi membran lambung [2]. dalam labu distilasi, dan 500 μl asam asetat ditambahkan. Sam-ples diekstraksi
Setelah asupan oral, penghambatan kemampuan psikofisik terjadi dengan distilasi uap menggunakan Unit Distilasi BÜCHI B-324; Laju aliran fase
dalam beberapa detik hingga 1 atau 2 menit. Dalam beberapa kasus, gerak adalah 0,7 mL / menit. Uap dipadatkan dan dikumpulkan dalam tabung
interval latensi 5-10 menit atau lebih telah dilaporkan. Segera setelah plastik 10 ml berisi 1 ml NaOH 1 M, hingga volume total 10 ml. Kemudian, 1 ml
kehilangan kesadaran, kejang-kejang, hipotensi, apnea, syok, dan, setiap ekstrak diencerkan lagi hingga 10 ml dengan NaOH 0,1 M (pengenceran
akhirnya, kematian akibat asfiksia seluler telah diamati [3]. akhir: 1:100 v/v).
Sianida adalah racun yang banyak digunakan untuk pembunuhan atau
bunuh diri. Di seluruh dunia, kasus keracunan sianida fatal dan non-fatal
baru-baru ini telah dilaporkan [4-10]. Di Seoul (Korea), 255 kematian 2.3 | Sample analysis
akibat keracunan cya-nide dijelaskan antara tahun 2005 dan 2010; Dari
Analisis dilakukan dengan Anion-Exchange Liquid Chromatography with
jumlah tersebut, 97,3% adalah bunuh diri. Rute administrasi yang paling
sering adalah lisan (98,8%) [11]. Pulsed Amperometric Detection (IC-PAD). Untuk tujuan ini, Ion

Sianida adalah racun yang relatif mudah didapat. Di alam, glikosida Chromatograph Metrohm 761 Compact IC (perangkat lunak Metrohm

sianogenik hadir di berbagai tanaman melepaskan hidrogen sianida MagIC Net) dengan kolom polystyrene-divinylbenzene Metrohm Metrosep

melalui hidrolisis. Amigdalin adalah sisi gliko sianogenik yang paling A Supp 10–100/4.0 dengan ukuran partikel hanya 4,6 μm dan detektor

umum; Itu terkandung dalam biji atau biji almond pahit, aprikot, persik, amperometrik Metrohm 945 Professional Detector Vario. Gelombang

ceri, dan prem. Hal ini juga hadir dalam daun Prunus laurocerasus (juga amperometrik karakteristik untuk sianida dan sulfida digunakan. Profil

dikenal sebagai Cherry Laurel) dan tanaman com-mon lainnya [12]. potensial: 0,0 V untuk 900 ms, diikuti oleh -0,5 V untuk 100 ms dan,
Ini digunakan sebagai fumigan, rodentisida, dalam produk pertanian [13] akhirnya, 0,0 V untuk 100 ms. Suhu detektor: 35.0 °C. Fase gerak: 0,1 M

dan dalam elektroplating perhiasan [14]. Sianida mudah tersedia di situs web larutan natrium hidroksida dengan EDTA. Laju aliran: 1 ml / menit. Volume
injeksi: 10 μl.
[15], terutama di apa yang disebut "Deep Web" dan "Dark Web" [16].
Selain itu, hidrogen sianida adalah produk yang mungkin dari proses
pembakaran: hidrogen sianida hadir dalam knalpot mesin pembakaran 2.4 | Method validation
internal, dalam asap rokok, dan dalam asap yang berasal dari fusi bahan
plastik yang mengandung nitrogen [17]. Validasi analitik dilakukan menurut Laudani et al. [18]. Itu dilakukan pada

Diagnosis keracunan sianida mematikan kadang-kadang bisa rumit. darah babi kosong dan pada sampel organ (otak, paru-paru, dan hati).
Validasi dilakukan pada babi karena, seperti diketahui, ini merupakan studi
Tujuan dari penelitian ini adalah a) untuk mengembangkan dan
pemodelan terbaik karena menunjukkan kesamaan anatomi dan fisiologis
memvalidasi metode analisis dengan Anion-Exchange Liquid
sev-eral dengan manusia [19]. Sampel isi lambung kosong disiapkan
Chromatography dengan Pulsed Amperometric Detection untuk
dengan menyeragamkan 10 g daging, 10 g sayuran, dan 30 ml HCl 0,1 M.
mendeteksi dan mengukur sianida dalam darah, dan, untuk pertama
Linearitas, akurasi, presisi, batas deteksi (LOD), batas bawah
kalinya dengan tech-nique ini, dalam kandungan lambung, dan dalam
kuantifikasi (LLOQ), interferensi matriks, dan carryover dinilai untuk setiap
sampel organ dan b) untuk melaporkan nilai cya-nide yang ditemukan
matriks yang diuji:
dalam dua kasus forensik nyata.

1. Linearitas dievaluasi dengan uji Mandel menggunakan kalibra- 6 titik-

2 | BAHAN DAN METODE kurva tion direplikasi lima kali (0,25-0,5-1,0-2,5-5,0-7,5 μ g / ml atau μ g
/ g);
2.1 | Bahan kimia dan reagen 2. Sensitivitas ditentukan oleh evaluasi batas deteksi (LOD) dan batas bawah
kuantifikasi (LLOQ). LOD dan LLOQ diukur dengan mengevaluasi rasio

Kalium sianida KCN (≥98,0%) dibeli dari Sigma Aldrich. Air ultramurni sinyal/noise (S/N) untuk setiap matriks.

(UHPLC-MS / MS), asam asetat glasial (≥99,5%), natrium hidroksida 0,1 M LOD ditetapkan pada konsentrasi dengan S/N > 3. Konsentrasi analit dengan

(≥99,9%), dan natrium hidroksida 1,0 M (≥99,9%) diperoleh dari Reagen S/N > 10 dipilih sebagai LLOQ; Selain itu, kami telah menetapkan
Carlo Erba. Garam EDTA disodium (≥98,0%) dibeli dari VWR Chemicals.
bahwa LLOQ harus kurang dari 0,26 μ g/ ml atau μ g / g, yaitu, lebih
Garam timbal sulfat (≥98,5%) diperoleh dari Thermo Scientific.
rendah dari konsentrasi terendah yang dilaporkan sebagai beracun [20];
ZUCCARELLO et al. | 1619

1. Presisi (sebagai standar deviasi relatif RSD%) dan akurasi (sebagai persen #2. Seorang pria berusia 45 tahun ditemukan tewas di rumahnya.
pemulihan R%) dihitung untuk dua tingkat konsentrasi (1,0 Tubuh pria itu ditemukan tergeletak di tanah. Wajahnya sianotik. Tenaga
dan 5,0 μ g/ml atau μ g/g) untuk setiap matriks. Enam alikuot dari masing- medis yang turun tangan menyatakan kematian karena sebab alami; Tidak

masing sampel diekstraksi dan dianalisis. Untuk darah, RSD% dan pemulihan ada deskripsi adegan yang dilaporkan. Pada awalnya, kematian itu

harus antara 80% dan 120% (yaitu, akurasi hingga 20%). Untuk or-gans dan dikaitkan dengan peristiwa jantung akut, meskipun pria itu dalam keadaan

kandungan lambung, karena kompleksitas matriks ini, ditetapkan bahwa sehat. Namun, beberapa bulan kemudian, kekasih sang istri diperiksa oleh

presisi harus hingga 30% sedangkan covery ulang harus antara 70% dan polisi. Dia melaporkan bahwa istri pria itu membunuh suaminya dengan

130% (yaitu, akurasi hingga 30%); meracuninya dengan minuman yang mengandung sianida dan warfarin.

2. Gangguan matriks dinilai menggunakan tiga sampel darah babi dan Bekas humasi tubuh dibuang; Organ dan kandungan lambung

organ kosong untuk memverifikasi bahwa tidak ada komponen lain dikumpulkan untuk analisis toksikologi. Kandungan lambung ditemukan

(yaitu, sul-fida), kecuali analit, yang berkontribusi terhadap hasil; kering dan berat totalnya adalah 650 g. Investigasi otopsi dan

• Carryover dinilai dengan enam analisis sam-ples kosong yang histopatologi-logis tidak menunjukkan penyakit kardiovaskular akut atau
kronis, serta fenomena hemoragik organ internal yang dapat
diekstraksi, mengikuti analisis sampel pada 5,0 μ g / ml atau μ g/g. menyebabkan kematian.
Untuk kedua kasus, analisis sianida dilakukan dalam waktu 15 hari setelah

pengumpulan sampel; Selain itu, penyelidikan toksikologi rutin dilakukan.

2.5 | Case reports
Selanjutnya, dalam kasus # 2, analisis LC-MS / MS warfarin dilakukan pada otak, hati,

ginjal, dan isi lambung.

#1. Seorang pria berusia 72 tahun ditemukan tewas di rumahnya, terbaring di
tempat tidur. Subjeknya adalah seorang pembuat jam; Dia dipengaruhi oleh
depresi dengan upaya bunuh diri sebelumnya. Sebuah surat ditemukan di meja
3 | RESULTS
samping tempat tidur di mana pria itu mengomunikasikan niatnya untuk bunuh
diri dengan sianida. Sebuah gelas ditemukan di atas saputangan, di mana 3.1 | Validation of method
tertulis "racun sianida"; Sebuah kantong plastik, dengan indikasi yang sama,
ditemukan di lantai. Pakaiannya tidak menunjukkan noda darah atau laserasi. Pada Tabel 1, untuk setiap matriks, rentang kalibrasi dan koefisien korelasi
Hakim hanya membuang pemeriksaan eksternal postmortem dan pengambilan 2
terkait (r ), LOD dan LLOQ dilaporkan. Semua kurva kalibrasi menunjukkan
sampel darah femur. Pada pemeriksaan eksternal, sianosis wajah, rigor mortis koefisien linier lebih tinggi dari 0,98.
awal, suhu tubuh 28 ° C (suhu eksternal 23 ° C), dan lividitas postmortem Kinerja metode diskema pada Tabel 2. Untuk setiap matriks, nilai rata-
merah ceri terdaftar; Tidak ada fenomena transformatif dan tidak ada lesi rata, standar deviasi, standar deviasi relatif RSD%, dan persen pemulihan R
traumatis yang hadir. Sisa pemeriksaan eksternal biasa-biasa saja. % telah dihitung.

2
TA B L E 1 Calibration ranges (n = 5) and Units n. replication Range r LOD LLOQ
2
related curve correlation coefficients r ,
LOD, and LLOQ Blood μg/ml 5 0.25–7.5 0.9915 0.05 0.13
Lung μg/g 5 0.25–7.5 0.9833 0.09 0.24
Brain μg/g 5 0.25–7.5 0.9886 0.08 0.21
Liver μg/g 5 0.25–7.5 0.9837 0.07 0.19
Gastric content μg/g 5 0.25–7.5 0.9844 0.07 0.17

TA B L E 2 Method performance
Spiked Standard
parameters at 1.0 and 5.0 μg/ml or μg/g Units concentration Mean Dev. RSD% R%

Blood μg/ml 1.0 0.92 0.13 14.1 92.0

5.0 4.63 0.69 14.9 92.6

Lung μg/g 1.0 0.79 0.19 24.1 79.0

5.0 4.35 1.20 27.6 87.0

Brain μg/g 1.0 0.82 0.19 23.2 82.0

5.0 4.45 0.82 18.4 89.0

Liver μg/g 1.0 1.09 0.28 25.7 109.0

5.0 4.71 1.15 24.4 94.2

Gastric content μg/g 1.0 1.15 0.26 22.6 115.0

5.0 4.76 0.98 20.6 95.2
1620 | ZUCCARELLO et al.

Precision and accuracy fulfilled the acceptance criteria (<30%) and gastric content (1.2 μg/g); warfarin was also found in brain (0.9 μg/g),
for all matrices, at each concentration. The RSD% ranged between liver (8.3 μg/g), kidney (6.6 μg/g), and gastric content (23.9 μg/g).
14.1% (blood) and 27.6% (lung), with a mean value of 21.6%. The R% Figures 3 and 4 show the chromatograms of blood sample of
ranged between 79.0% (lung) and 115.0% (gastric content), with a case #1 and brain sample of case #2, respectively. The cyanide con-
mean value of 93.5%. centrations found in samples of both real cases are summarized in
Extracted blank samples showed no matrix interference and no Table 3. In both cases, no other substances of toxicological interest
cyanide concentration above LODs (Figures 1 and 2). were found.

3.2 | Case reports 4 | DISCUSSION

Case #1: a blood cyanide concentration of 0.99 μg/ml was detected. Case The analytical method reported here, satisfied all acceptance criteria
#2: cyanide was found in brain (1.3 μg/g), lung (0.8 μg/g), liver (1.6 μg/g), for validation. The RSD% ranged between 14.1% (blood) and 27.6%

FI G U R E 1 Chromatogram of cyanide
analysis on blank brain sample [Color
figure can be viewed at wileyonlinelibrary.
com]

FI G U R E 2 Chromatogram of
cyanide analysis on blank blood sample

FI G U R E 3 Chromatogram of cyanide
analysis on blood sample of case #1
[Color figure can be viewed at
wileyonlinelibrary. com]
ZUCCARELLO et al. | 1621

FI G U R E 4 Chromatogram of cyanide
analysis on brain sample of case #2 [Color
figure can be viewed at wileyonlinelibrary.
com]

TA B L E 3 Cyanide concentrations found in samples of investigation to ascertain death from cyanide poisoning in those
both examined cases cases where biological fluids are not available, as in our case #2.

Blood Brain Lung Liver Gastric The sample extraction reported here is different than that de-

Case (μg/ml) (μg/g) (μg/g) (μg/g) content (μg/g) scribed by Sim et al. Our procedure of distillation in acid environ-
ment was developed from the classic one reported by Gettler and
#1 0.99 / / / /
Golbaum [24]. Unlike to Sim et al. opinion, a total cyanide detection
#2 / 1.3 0.8 1.6 1.2
gives useful information on the amount of cyanide present in the
body at the time of death. These data are very important especially
(lung) with a mean value of 21.6%. The R% ranged between 79.0% when death occurs slowly or when there is a long interval of time
(lung) and 115.0% (gastric content) with a mean value of 93.5%. between death and autopsy.
Blood analysis showed the best precision and accuracy for both ex- We agree with Sim et al. to consider the chromatographic tech-
amined concentrations. niques (GC), which require cyanide derivatization, too problematical and
The use of internal standard would be useful to improve preci- expensive; moreover, if the analyzed matrix is more complex than blood
sion and accuracy, especially for organs and gastric content. (as, for example, organs) this approach could be low accurate.
However, there is no compound that can be used as internal In the present study, we described and validated a simple, rapid,
standard in this technique [21]. and inexpensive method. It can be used both to assess a cyanide
The method showed a good selectivity and sensitivity. No car- acute fatal intoxication and, without the dilution here used, to detect
ryover and any matrix interferences were detected. The extraction low concentrations due to environmental or smoke exposure.
method in acid environment by steam current distillation reduced Cyanide does not always have lethal effects, depending on the
matrices interferences and allowed to carry out the analysis with concentrations it reaches in the body. Discriminate lethal from no--
good precision and accuracy. lethal cyanide concentrations is important in forensic investi-gation.
Anion-Exchange Liquid Chromatography with Pulsed Amperometric In active smokers, cyanide concentrations are generally 20– 100
Detection proved to be an excellent technique for cy-anide and sulfide times lower than fatal ones. Blood cyanide concentrations of healthy
separation as shown in Figure 4. As the amperomet-ric wave used here non-smokers ranged from 0.0027 to 0.0102 μg/ml [25] In smokers,
is characteristic for the detection of cyanide and sulfide, the blood cyanide concentrations ranged from 0.00962 μg/ml to 0.0208
chromatographic separation of these two compounds was crucial to μg/ml; no data on human organs of the general population or
evaluate the resolution and the selectivity of this technique. smokers were reported [26]. In our cases, cyanide concentrations
Although various methods to identify and quantify this sub- were higher than those normally present both after a common envi-
stance are reported in the literature, as colorimetry, spectropho- ronmental and/or food exposure, and in smokers.
tometry, near-infrared spectroscopy, electrochemical detection, In case #1, blood cyanide concentration was high enough to cause
chromatography coupled with mass spectrometry, capillary electro- lethal poisoning. In case #2, although blood was not available (the corps
phoresis, fluorometry, chemiluminescence, and atomic absorption was exhumated 1 year after death), the examined samples resulted alike
spectrometry, they have been mainly applied to biological fluids (i.e., useful. Indeed, organs cyanide concentrations were close to those
blood, urine, and saliva) [22]. reported in other fatal cases. For example, Musshoff et al. reported the
Sim et al. (2021) [21] and Jaszczak et al. (2017) [23] applied this following cyanide concentrations after HCN in-halation for suicidal intent:
method to blood, urine, saliva, and sweat. In our study, the IC-PAD 5.3 μg/ml in blood, 0.96 μg/g in brain, 2.79 μg/g in lungs, and 0.20 μg/g in
technique was also validated and applied to organs and gastric con-tent, gastric content [1]. In fire vic-tims, Htike et al. have observed the
in addition to blood matrix. Its application to unconventional matrices, as following cyanide mean values: 0.06 μg/g in brain, 2.2 μg/g in lung, 0.08
organs and gastric content, is the only option in forensic μg/g in liver, and 0.09 μg/g in
1622 | ZUCCARELLO et al.

kidney [27]. Particularly, in case #2, brain and liver cyanide concen- In conclusion, the method reported here showed a good selec-
trations resulted higher than those reported in the literature, while tivity and sensitivity with no carryover and matrix interference. The
cyanide lung concentration was lower. This difference is explainable extraction by steam distillation in acid environment reduced the in-
considering that the cases of poisoning reported by Musshoff et al. terference of the matrices and allowed to perform the analysis with
[1] and Htike et al. [27] are related to HCN inhalation, while our case good precision and accuracy.
has occurred after ingestion. Finally, the cyanide concentrations found in the two reported
Cyanide concentration found in gastric content of case #2 re-sulted cases, much higher than those normally present in the body, must
lower than those reported in a review by Rhee et al. [28]. In the 21 be considered suitable to cause the death by poisoning.
described cases related to cyanide intoxication by oral ingestion, the
authors reported that gastric content cyanide concentrations ranged from ACKNOWLEDGEMENTS
2.0 to 6398.0 mg/kg. Neither the doses taken by the subjects, nor the Supported by Prof. Margherita Ferrante, Director of the
amounts of gastric content have been described. The authors observed Environmental and Hygiene Laboratories of University of Catania.
that no correlation was found between gas-tric content and blood Open Access Funding provided by Universita degli Studi di Catania
concentrations [28]. As they were all cases of suicide, it is possible to within the CRUI-CARE Agreement.
hypothesize that the subjects have in-gested a large amount of cyanide
to be sure of dying. However, the concentrations that can be found in ORCID
gastric content could not have toxicological relevance if the taken dose Pietro Zuccarello https://orcid.org/0000-0002-7425-0032
and the total amount of gastric content are not known. In fact, for the Cataldo Raffino https://orcid.org/0000-0001-6212-2570
same ingested dose, the gastric substance concentration will be higher if Nunziata Barbera https://orcid.org/0000-0002-9157-5947
the stomach is almost empty, compared to when it is full. It is our opinion
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