Simultaneous Screening for 45 Poisonous Alkaloids in Blood by LC-MS/MS

ZHAI Jin-xiao,SHEN Min,LIU Wei

（1.Department of Biochemistry and Molecular Biology,Medical College of Soochow University,Suzhou 215123,China;2.Shanghai Key Laboratory of Forensic Medicine,Institute of Forensic Science,Ministry of Justice,P.R.China,Shanghai 200063,China）

Introduction

Alkaloidsareorganiccompoundswithbasic chemical properties,containing at least one nitrogen atom normally in a heterocyclic ring,and occurring chiefly in vascular plants and some fungi[1],and about 10%to 25%of higher plants contain alkaloids.Furthermore,differenttissuesofthesame plantsmaycontaindifferent alkaloids;mostare weak bases,but some such as theobromineand theophylline,are amphoteric[2].Alkaloids with various pharmacologicaleffectsareusedasmedications;however,many are toxic to other organisms.Such alkaloids may act via various pharmacological mechanisms,e.g.activation or blocking of receptors or ion channels,causing severe or even lethal poisoning[3].Besides cardiac glycosides,the alkaloids most frequently involved in plant poisonings were aconitine,atropine,colchicine,nicotine,physostigmine,and scopolamine.Alkaloids poisoning can occur as a result of overdose,incorrect processing and preparation,contamination,misidentification,and also in homicidal and suicidal cases.In case of abuse or intoxication,detectionandquantificationofpoisonous alkaloids in the biological materials is necessary in clinical and forensic toxicology[4].

So far several methods for the analysis of different kinds of alkaloids in biological samples have beenreported,suchashighperformanceliquid chromatography（HPLC）[5],gas chromatography-mass spectrometry（GC-MS）[6-10]and liquid chromatography-tandem mass chromatography（LC-MS/MS）[11-13].Hyphenated mass spectrometric techniques,particularly LC-MS/MS,provide sensitivity,selectivity and specificity necessary for the screening analysis of poisonous alkaloids.LC-MS/MS with multiple-reaction monitoring（LC-MS/MS-MRM）appears to be a superduper method for analyzing complex and poorly volatile alkaloids in blood,because of the presence of a nitrogen ring and the relatively low concentrations to be measured[14].However,most of these approaches detect only one particular group of alkaloids,or at most several alkaloids of different classes.It is necessary to have a method of simultaneous detection for a broad range of toxic plant alkaloids with different groups in biological samples in investigating suspected alkaloids poisonings,especially in the cases of medications unknown from the medical histories.

The objective of the current study was to develop a sensitive and selective LC-MS/MS method for a simultaneous detection of multiple different types of 45 poisonous alkaloids in blood.The proposed method,validated by assessing selectivity,limit of detection（LOD）,limits of quantification（LOQ）,linearity,accuracy and precision,was successfully applied to the analysis of real forensic samples of suspected alkaloids poisonings.

Materials and methods

Ch emicals and reagents

Ephedrine,physostigmine,ergometrine,strychnine,atropine,scopolamine,tetrahydropalmatine,pi-locarpine,hypaconitine,mesaconitine,bulleyaconitine,aconitine,lobeline,theophylline,brucine,colchicines,cocaine,homatropine,codeine,papaverine,narcotine,homoharringtonine,galanthamine,thebaine,oxycodone,piperine,bullatine,oxymarine,matrine,reserpine,diprophylline,camptothecin,morphine,sinomenine,barbering,jatrorrhizine,palmatine,protopine,tubocurarine,huperzine,sophoridine,hanfangchin,theobromine,anisodamine,hydroxycamptothecin and SKF525Aas internal standard（IS）from the National Institute for the Control of Pharmaceutical and Biological Products（Beijing,China）were prepared at 1 mg/mL in methanol or water as a stock solution,respectively.A working solution of SKF525Awas prepared at 0.5 μg/mL in methanol added to the samples.The working solutions of each analyte were prepared by independentdilutionfrom each stock solution according to their LOQ and preparation of the corresponding concentrations of mixed standards.All working solutions were stored at 4℃during the validation.

Of HPLC grade,acetonitrile,methanol,diethyl ether,ethyl acetate were obtained（Sigma-Aldrich Corporation,USA）;ultra-pure water,by Milli-Q Element Ultrapure Water System（Millipore Corporation,USA）;buffer solution（pH=6.8,pH=9.2）;blank blood with citrate-anticoagulated,from Shanghai Blood Center.Other reagents were all of analytical-reagent grade.

Ex traction procedures

The extraction procedures were based on liquid-liquidextractionwithpH=9.2buffersolution and diethyl ether.To 1 mL of blood sample was added 1 mL of pH=9.2 buffer solution by 3-min vortex,followed by an addition of 3 mL of diethyl ether to extract alkaloids and IS from the blood samples by 3-min shake.The organic phase was separated from the aqueous phase through a centrifugation at 2 500 r/min for 3 minutes.The supernatant was transferred and evaporated to dryness under a stream of nitrogen at 50℃.Then 100 μL of mobile phase［V acetonitrile:V ammonium acetate（20 mmol/L）=7:3］was added to the residue and reconstituted via 3-min shaking.The solution was transferred to autosampler vials and 10μL each was injected into the LC-MS/MS systems.

LC -MS/MS procedure

LC was performed using a system composed of an AcquityTMUltra Performance LC（Waters Corporation,USA）,a quaternary pump,an online degasser,and an autosampler.Chromatographic separation was achieved with a Capcell Pak C18（250 mm×2.0 mm,MGⅡ,5μm）protected by an end-capped C18guard column（12.5mm×2.1mm,5 μm）,at room temperature.The injection volume was 10 μL with a total run time of 12 minutes.And the LC mobile phase was used to separate the analytes with a composition of 70%acetonitrile and 30%buffer solution（20 mmol/L ammonium acetate and 0.1%formic acid,pH=4）,which was run at an isocratic elution,the total flow rate through the column being 200μL/min.

MS/MS detection was performed on an MDS Sciex API 4000 Q trap mass spectrometer（Applied Biosystems,USA）with an Ion Max ESI probe.The instrument was operated in the positive electrospray ionisation mode with an ion spray voltage of 5500V and source temperature at 500℃.The collision gas was nitrogen as medium,the curtain gas 30 psi;the nebulising gas（GS 1）,40 psi,and the turbo spray gas（GS 2）50 psi.Multiple-reaction monitoring（MRM）technology was used to detect the parameters of alkaloids.

Me thod validation

The current method was validated according to the suggestions by Peters et al.[15],the validation data oftheanalyticalmethodincludingcalibration curve,precision,accuracy,sensitivity and specificity.Calibration curves were evaluated by spiking blank blood samples with target alkaloids at 7 concentration levels,respectively.The LOD on blood was evaluated by decreasing the concentrations of alkaloids until a response equivalent to three times the background noise was observed.And LOQ were determined from the repeatability and accuracy（S/N≥10）for blood.The selectivity of the method was investigated by analyzing 10 blank blood samples from different sources.A blank sample spiked with IS was analyzed to check for absence of analyte ions in the respective peaks of the IS.The quality control（QC）samples were prepared at three concentration levels,low,middle and high.The accuracy and precision of intra-day（n=8）and inter-day（n=24）were evaluated through the replicate analysis of QC samples on the same day and 3 consecutive days,respectively.The intra-day and inter-day precisions were expressed as relative standard deviation（RSD,%）,and accuracy was determined as bias.

Results and discussion

Op timization of the extraction conditions

The standard liquid-liquid extraction（LLE）procedure is usually used for extraction of alkaloids from biological materials.Compared with most other classes of natural compounds,alkaloids are characterized by a great structural diversity with no uniform classification of alkaloids[16].Most alkaloids are weak bases,but some are amphoteric.Since the chemical properties of these alkaloids are different,the effect of different pH on the recovery of alkaloids was examined in the current method.As a result of the comparison,most alkaloids at pH=9.2 were better for the pH adjustment of the extraction conditions.The peak areas were measured for LLEusing different organic solvents,and for the comparison of two extraction solvents,diethyl ether or ethyl acetate.And a better recovery was achieved for LLE with ethyl acetate for the most alkaloids.Butethylacetateevaporateddifficultlyandwas prone to blood esterification phenomenon.The results showed that extraction efficiency of diethyl ether was also more than 50%;therefore,it was selected as the extraction solvent and pH=9.2 buffer solution was used for the pH adjustment of the extractionconditions.Insuchalkaloidsasjatrorrhizine,huperzine and hydroxycamptothecin,the extractionefficiencieswerefoundtobelessthan 50%,which could be explained by their unique chemical properties.But they could apply to this extraction condition for qualitation and quantitation on account of their good reproducibility.

In conclusion,the described extraction procedure resulted in a high sensitivity of most alkaloids and little matrix interference,thus being considered appropriate for most analytes.

Method validation

Se lectivity

The blank blood samples were analyzed,and no interfering peaks were observed at the retention time characteristic of these alkaloids in the blood matrix,indicating the credible and acceptable selectivity of the method（Fig.1）.A blood sample spiked with the target compounds at LOQ level demonstrated instrument sensitivity.Under the optimized chromatographic conditions,most of the analytes were well separated.Hanfangchin and piperine were not well separated,most probably because the liquid phase conditions were not the most suitable to them.Having a good reproducibility,however,they could be used for qualitative and quantitative analysis.

Li nearity,LOD and LOQ

MS/MS parameters,regression equation,linear range,r,LOD and LOQ for 45 alkaloids and IS（SKF525A）determination were listed（Table 1）,where it was shown that using tandem MS,the assay was linear from 0.1 to 4000ng/mL for 45 alkaloids,respectively.The calibration curves（analyte peak area/IS peak area for the Y-axis and analyte concentration for the X-axis）were assessed through least-squares linear regression fit（y=ax+b）.As indicated by the regression equations of 45 alkaloids（Table 1）,their LOD ranged from 0.05 to 25 ng/mL in blood,and the coefficients of determination（r）,from 0.995 1 to 0.999 4.The LOD values were determined by spiking whole blood samples with an appropriate.And ion suppression,caused by matrix,could affect the LOD values[17].The LOQ ranged from 0.1 to 50ng/mL in blood.The LOQ of anisodamine and hydroxycamptothecin were relatively high,because of instability of some objective factors and inapplicability of their unique chemical properties in the current method.

Fig.1MRM chromatograms

Table 1MS/MS parameters,regression equation,linear range,r,LOD and LOQ for 45 alkaloids

Continued

Continued

Ac curacy and precision

Accuracy and precision of intra-day and inter-day were evaluated based on the replicate analysis of QC samples on the same day and 3 consecutive days,respectively.As a result,the data for the accuracy andprecisionwerewithintherequired limits.The accuracy in human blood ranged from 85.9%to 114.95%for 45 alkaloids at different QC levels,and the intra-day and inter-day precision of the tests were between 2.01%to 14.77%,showing that good precision could be obtained using the current method.Therefore,the approach could be accurate and reliable through the precision and accuracy experiments.

In the assays,the worst RSD of determination,foundinbarbering,diprophylline,hydroxycamptothecin,oxymarine,palmatine,reserpine,tubocurarine,was most probably due to the instability of theirchemicalproperties,efficiencyoftheion source and matrix effects of impurities on the column efficiency.

The difficulties could be mainly attributed to threefeaturesinanalyzing thealkaloidspresent:Most of the alkaloid skeletons lack a conjugated system;isomericandstereoisomericalkaloidsare generally present,andtheidentificationof chromatographic peaks on the basis of retention times under the chromatographic conditions employed can be ambiguous;and the levels of some minor alkaloids are extremely low,necessitating the use of a highly sensitive detector[18].In the current method,however,such analytical problems were overcome using the LC-MS/MS method.

Application

Case1:A37-year-oldmalewasprescribed some Chinese traditional medicine for his injured right foot.Having taken it orally,he felt unwell and went to the hospital.Two hours later,he died.From an autopsy,the biological samples of blood,urine,gastric content were collected.The general toxicological screening and alkaloids analysis were requested,the results showing that bulleyaconitine was detected in the biological samples using the current method.

Case 2:A 47-year-old male died after drinking home-made medicinal liquor.His vomits,blood andmedicinal liquor samples were collected.The current method was applied to the analysis of alkaloids in the biological specimens.The results showed the detection of aconitine in the biological samples,aconitine concentration in blood and medicinal liquor 7.6ng/mL and 129.5μg/mL,respectively.

Conclusion

In the current study,LLE was chosen to extract target substances,and a sensitive LC-MS/MS targetscreening,identificationandquantification method for 45 alkaloids in blood was developed and validated.Under the optimized LC-MS/MS conditions described,all of the analytes were well separated within 12 minutes.Thanks to the current method,a broad range of toxic plant alkaloids of different groups in blood were detected,with LOD of most alkaloids less than 1 ng/mL.Although the precision of certain substances was poor because of the instability of some objective factors,the approach was extremely useful as a preliminary（pilot）method in screening for alkaloids in blood.The method can be readily expanded to encompass other biological samples,if necessary,for it was successfully applied to the real cases of suspected herbal poisonings.

Acknowledgements

This current study was funded by the Council of Shanghai Key Laboratory of Forensic Medicine（14DZ2270800）and grants from Ministry of Finance,P.R.China（GY 2012-G2）.

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