Applica ions o Tandem Mass Spec ome y (LC–MSMS) in es ima ing he pos -mo em
in e al using he biochemis y o he i eous humou
Elena Lendoi oa, C is ina Co dei ob,c, Ma ía S. Rod ıíguez-Cal od, Dua e N. Viei ab,c,
José M. Suá ez-Peña andad, Manuel López-Ri adullaa,d, José I. Muñoz-Ba úsa,d,*
a Ins i u e o Fo ensic Science, Uni e si y o San iago de Compos ela, Spain
b Na ional Ins i u e o Legal Medicine and Fo ensic Sciences o Po ugal – Cen e B anch,
Po ugal
c Cen e o Fo ensic Sciences (CENCIFOR), Uni e si y o Coimb a, Po ugal
d Depa men o Pa hology and Fo ensic Science, Uni e si y o San iago de Compos ela, Spain
*Co esponding au ho : joseignacio.munoz.ba [email protected]
ABSTRACT
I is widely accep ed ha he quan ifica ion o po assium concen a ions ([K+]) and hypoxan hine
([Hx]) in he i eous humou is use ul in es ima ing he ime o dea h wi hin a ecen ime
in e al. Despi e all he ad ances made in his a ea, i is well ecognized ha di ficul ies in
calib a ion, alida ion and he use o diffe en me hodologies and ins umen a ion om diffe en
labo a o ies may lead o ob aining diffe en concen a ions om he same sample. The
ex ac ion o he i eous humou i sel should also be ca ied ou wi h a p ecise echnique,
aking ca e du ing he p ocedu e o a oid causing any ascula inju y ha migh lead o haema ic
con amina ion. Any fluid ha is no clea could lead o e oneous esul s and should be
disca ded. We p esen a new LC–MSMS me hod de eloped o quan i a i e and quali a i e
analysis o [Hx] ( alid also o guanine and xan hine), and quali a i e de e mina ion o u ic acid
in i eous humou . We also in oduce a me hodology o assess haema ic con amina ion in o de
o imp o e he es ima ion o ime since dea h. The me hod was ully alida ed in e ms o
linea i y, sensi i i y, imp ecision, analy ical eco e y, ex ac ion and p ocess e ficiency and
ma ix effec .
Keywo ds: Tandem mass spec ome e ; Pos mo em in e al; Hypoxan hine; Vi eous humou
1. In oduc ion
In he field o o ensic pa hology one o he mos impo an issues is he co ec es ima ion o
he pos mo em in e al (PMI). This is o pa icula ele ance in c iminal in es iga ion, whe e
ecou se o a me hod ha p o ides a PMI whose p ecision can be upheld in a cou o law can
be c ucial [1,2]. I is widely accep ed ha he quan ifica ion o po assium concen a ions ([K+])
and hypoxan hine ([Hx]) in he i eous humou is use ul in es ima ing he ime o dea h wi hin
a ecen ime in e al [3–7].
The impo ance o his p oblem is demons a ed by he ac ha i has become he subjec o
nume ous publica ions in leading jou nals in his field, which highligh s he di ficul ies o his
app oach, and has led o u he compa a i e s udies [8]. I has been epo ed ha u ea (U)
may modi y he ela ionship be ween he concen a ion o i eous po assium and PMI [4,9,10]
and ha he cause o dea h i sel can also modi y his ela ionship [10,11]. Diffe en analy ical
me hods o quan ifica ion o po assium and [Hx] ha e been de eloped in o de o imp o e
accu acy in es ima ing he ime o dea h [12,13].
Al hough an in e se p edic ion by changing he a iables is he s a is ical me hod o choice
when using linea eg ession o ela e he [K+] o [Hx] in he i eous wi h PMI [4,14], o he
mo e flexible me hods o eg ession ha e been de eloped which by including all he possible
a iables [K+], [Hx], [U], in he i eous humou oge he wi h he cause o dea h, p o ide a
mo e accu a e es ima e [11]. Al hough he calcula ion is much mo e complex han he simple
applica ion o he ypical single-line eg ession o mula, a ee o use s a is ical package has
been p esen ed which p o ides a as and in ui i e esul [15].
Despi e all he ad ances made in his a ea, i is well ecognized ha he di ficul ies in
calib a ion, alida ion, diffe en me hodologies and ins umen a ion om diffe en labo a o ies
may lead o ob aining diffe en concen a ions om he same sample [16,17]. I has also been
demons a ed ha p e- ea men o samples be o e analysis may lead o some a ia ions in
he de e mina ion o po assium and u ea [17]. Howe e , no only p e- ea men o he sample,
bu he ex ac ion o he i eous humou i sel should be ca ied ou wi h a p ecise echnique,
aking ca e du ing he p ocedu e o a oid causing any ascula inju y which migh lead o
haema ic con amina ion. Any fluid ha is no clea could p oduce e oneous esul s and should
be disca ded [2,4,9,18].
Liquid ch oma og aphy coupled o mass spec ome e (o andem mass spec ome e ) (LC–MS
o LC–MSMS) has ecen ly become inc easingly impo an in pos -mo em oxicology as a
eliable echnique o ou ine analysis. Unlike gas ch oma og aphy coupled o mass
spec ome e (o andem mass spec ome e ) (GC–MS o GC–MSMS), i enables he analysis o
pola compounds wi h no de i a iza ion s ep, bu has he same sensibili y and specifici y as
GC–MSMS [19]. LC–MSMS has been employed o he iden ifica ion and/o quan ifica ion o
d ugs o abuse, he apeu ic d ugs, poisons and/o hei me aboli es in pos -mo em samples
such as whole blood, plasma, se um, u ine, ce eb ospinal fluid, i eous humou , li e and hai
[20].
The aim o his p esen pape is o imp o e he es ima ion o PMI wi h i eous biochemis y
using a new analy ical me hod, based on LC–MSMS, o he de e mina ion o Hx in i eous
humou ( alid also o u ic acid (UA), guanine (G), and xan hine (X)), and a me hodology o
assess haema ic con amina ion in appa en ly clea samples o i eous humou showing no
haema ic colo a ion due o he low p esence e y h ocy es in he sample.
2. Ma e ials and me hods
2.1. Chemical and eagen s
Hx, G, X and UA as a solid o m we e pu chased by Ac os O ganics (Geel, Belgium). The in e nal
s anda d (IS), 5-(p-me hylphenyl)-5-phenylhidan oin, was supplied by EGA-Chemie (S einheim,
Ge many). Ace oni ile and me hanol we e p o ided by Pan eac Quimica S.A.U. (Ba celona,
Spain). Ammonia solu ion (32%) and o mic acid we e supplied by Scha lau (Sen mena , Spain).
Ammonium ace a e was supplied by Pan eac (Ba celona, Spain). Pu ified wa e was ob ained in
he labo a o y using a Milli-Q sys em (Le Mon -su -Lausanne, Swi ze land). OASIS MAX ca idges
(60 mg, 3 mL) we e om Wa e s (Mild o d, USA).
2.2. Ins umen a ion
The HPLC sys em consis ed o a Wa e s Alliance 2795 Sepa a ion Module wi h a Wa e s Alliance
se ies column hea e /coole (Wa e s Co p, Mil o d, USA). Fo he ch oma og aphic sepa a ion,
an A lan is T3 (2.1 mm 100 mm, 3 mm) (Wa e s Co p, Mil o d, USA) analy ical column was used
a 30 8C. The mobile phase was ammonium ace a e 10 mM (pH = 4.5) (A) and ace oni ile (B),
wo king in g adien mode a a flow a e o 0.25 ml/min, and pu ge liquid was ace oni ile. The
g adien applied was as ollows: 0–2 min 100% A; 2–2.5 min om 100% o 20% A; 2.5–6.5 min
20% A; and 6.5–7 min om 20% o 100% A. The o al un ime was 14 min. Fo de ec ion, a
Qua o Mic oTM API ESCI iple quad upole (Wa e s Co p, Mil o d, USA) was employed. The
ins umen was ope a ed in elec osp ay in posi i e mode (ESI+) o p oduce p o ona ed
molecules o he analy es unde he ollowing op imized se ings: capilla y ol age 3.0 kV; sou ce
block empe a u e 130 8C; desol a ion gas (ni ogen) empe a u e 400 8C; desol a ion gas
flow a e 800 L/h; and cone gas (ni ogen) flow a e a 50 L/h.
Da a we e acqui ed in MRM (mul iple eac ion moni o ing) mode, which consis s in he selec ion
o he ion o in e es ( he p ecu so ) in he fi s quad upole (Q1), i s agmen a ion o o m
diffe en p oduc ions in he collision cell and finally he selec ion o a specific p oduc ion in he
hi d quad upole (Q3). The associa ion be ween he p ecu so and one p oduc ion is e e ed
as a ansi ion. T ansi ions, cone ol age and collision ene gy we e op imized by in usion o each
indi idual analy e in o he mass spec ome e (10 mM in NH4OH 2 mM) a 20 mL/min. Two
p ecu so -p oduc ansi ions pe compound we e moni o ed, excep o IS, o which only one
ansi ion was selec ed. In Table 1, MRM ansi ions, cone ol age, collision ene gy and e en ion
ime (RT) a e indica ed o each analy e and IS. Da a acquisi ion was con olled using MassLynx
4.0 so wa e and p ocessed wi h QuanLynx 4.0 so wa e (Wa e s Co p, Mil o d, USA).
2.3. Calib a o s and quali y con ol p epa a ion
The p ima y s anda d solu ion o each compound was p epa ed sepa a ely a e being weighed
in solid o m and econs i u ed in ammonia solu ion (32%); ini ial concen a ions o Hx, G and X
we e 10 mM, and 5 mM o UA. Each solu ion was p epa ed daily. Wo king s anda d solu ions
we e p epa ed in wa e by dilu ion o p ima y solu ions, and final concen a ions we e 1000, 200
and 100 mM o Hx and UA, and 100, 20 and 10 mM o G and X. The p ima y IS solu ion was
p epa ed in me hanol a 10 mM, and he wo king IS solu ion was p epa ed by dilu ion in
me hanol a 1 mM. Calib a ion cu es (10–400 mM o Hx and UA, and 1–40 mM o G and X)
we e p epa ed by adding 15, 30 and 60 mL o he co esponding wo king solu ion, and 25 mL o
wo king IS solu ion (1 mM). The analy es we e endogenous compounds, consequen ly a iable
concen a ions we e ound in con ol i eous humou ; o his eason calib a ion cu es we e
p epa ed in 150 mL o wa e .
Quali y con ols a low (10 mM o [Hx], 1 mM o G, 2 o X, and 20 o AU), medium (50 mM o
Hx and UA, and 5 mM o G and X) and high (150 mM o Hx, 25 mM o G and X, and 250 mM
o UA) concen a ions we e p epa ed daily in wa e om wo king solu ions sepa a e om hose
used o calib a o s.
2.4. Sample p epa a ion
Vi eous humou samples we e ini ially cen i uged o 10 min a 14,500 pm in a Mini Spin Plus
(Eppendo , Hambu g, Ge many). 2 mL o ammonium hyd oxide 2 mM and 25 mL o IS we e
added o an aliquo o 150 mL o i eous humou , p e iously cen i uged. Samples we e
submi ed o a solid-phase ex ac ion wi h OASIS MAX ca idges (Wa e s, Mild o d, USA). A e
condi ioning wi h 2 mL me hanol and 2 mL wa e , samples we e applied di ec ly in o he SPE
column. Cleanup was accomplished by sequen ial washes wi h 2 mL 5% ammonium hyd oxide
in wa e and 2 mL 5% ammonium hyd oxide in me hanol. Ca idges we e d ied o 10 min unde
acuum. Elu ion was pe o med in wo sequen ial s eps: fi s elu ion wi h 2 mL 2% o mic acid in
wa e , and second elu ion wi h 2.5 mL o mic acid in me hanol. Elua es we e collec ed in he
same ube and e apo a ed o d yness wi h ni ogen a 40 8C. Finally samples we e
econs i u ed in 100 mL ammonium ace a e 10 mM (pH = 4.5), and 30 mL we e injec ed in o LC–
MSMS.
2.5. Valida ion
The me hod was submi ed o a ull alida ion, including linea i y, limi o de ec ion (LOD), limi
o quan ifica ion (LOQ), imp ecision, analy ical eco e y, ex ac ion e ficiency, p ocess e ficiency,
and ma ix effec . Selec i i y was no e alua ed because i was no possible o ob ain blank
i eous humou ma ixes (i.e. wi hou Hx, G, X and UA).
Linea i y was de e mined by he analysis o ou calib a ion cu es p epa ed on 4 diffe en days.
These calib a ion cu es we e p epa ed in wa e , and leas -squa es eg ession was applied.
Accep able linea i y was achie ed i coe ficien o de e mina ion ( 2) was a leas 0.99, and
calib a o esidual was 20% a he LOQ and 15% a he o he concen a ion le els.
The LOD was defined as he lowes concen a ion wi h accep able ch oma og aphy, and he
p esence o he wo ansi ions wi h a signal- o-noise a io o a leas 3. The LOQ was he lowes
concen a ion wi h a signal- o-noise o a leas 10, imp ecision lowe han 20%, and analy ical
eco e y be ween 80% and 120%.
Imp ecision and analy ical eco e y we e de e mined a low, medium and high QC le els, wi h 5
eplica es on 4 diffe en days (n = 20), p epa ed in wa e . Imp ecision, exp essed as coe ficien
o a ia ion (CV) o he measu ed alues, was expec ed o be less han 15%. K ouwe and
Rabinowi z guidelines [21] we e ollowed o calcula ion o pooled in a-day, in e -day and o al
imp ecision. The analy ical eco e y was e alua ed as he pe cen age o he a ge concen a ion
(n = 20), wi h an accep ance c i e ion o 85–115%.
Ex ac ion e ficiency, p ocess e ficiency and ma ix effec we e de e mined using eal specimens
o i eous humou a wo le els o concen a ion (low and high) o Hx, G and X; and a one le el
o concen a ion o UA and IS. Howe e , analy es a e endogenous compounds, which a e
p esen in significan concen a ion in i eous humou specimens. Fo his eason, specimen
concen a ions we e p e iously measu ed (‘‘base alue’’); and hese ‘‘base alues’’ we e
sub ac ed om he alues o he same samples a e he addi ion o 20 o 60 mM o Hx, 100
mM o UA, and 2 o 6 mM o G and X (‘‘spiked alue’’). Ex ac ion e ficiency was calcula ed

compa ing he alue ob ained a e sub ac ion o a e age peak a eas o ‘‘base alue’’ and
‘‘spiked alue’’ o i eous humou specimens o ified p io o ex ac ion (n = 6) wi h hose
ob ained a e sub ac ion in specimens o ified a e ex ac ion (n = 6) a he same
concen a ion. P ocess e ficiency was de e mined by compa ing he alue ob ained a e
sub ac ion o a e age peak a eas o ‘‘base alue’’ and ‘‘spiked alue’’ o i eous humou
specimens o ified p io o ex ac ion (n = 6), wi h peak a eas o samples a he same nominal
concen a ions p epa ed in ini ial mobile phase (‘‘unex ac ed samples in mobile phase’’). Ma ix
effec was assessed by compa ing analy e peak a eas a e sub ac ion o ‘‘base alues’’ in 6
diffe en i eous humou ex ac ed samples o ified a e ex ac ion wi h analy e peak a eas o
‘‘unex ac ed samples in mobile phase’’.
Good quan ifica ion o eal samples wi h calib a ion cu es p epa ed wi hou ma ix was es ed
by he analysis o fi e i eous humou s be o e and a e adding 100 mM o Hx and UA, and 10
mM o G and X. The diffe ences be ween hese wo measu es we e e alua ed in e ms o
p ecision (calcula ed as %CV) and accu acy (exp essed as inaccu acy and as he pe cen age o
he mean ela i e e o (%MRE)).
2.6. E alua ion effec o blood con amina ion
Acciden al punc u e o he blood essels in he i eous humou du ing sample collec ion can
al e [Hx] alues, leading o an e oneous PMI in e p e a ion. To es he effec o blood
con amina ion, eal specimens we e con amina ed using ising blood concen a ions and he
esul s compa ed wi h blood- ee ma ix om he same sou ce. To simula e blood
con amina ion, a blood dilu ion a 5% was p epa ed in saline solu ion, and 1, 2, 3, 4, and 5 mL o
his dilu ion we e added o diffe en aliquo s (150 mL) o he same i eous humou . These
esul s we e compa ed wi h he alues ob ained om he same uncon amina ed i eous
humou . This me hodology was applied o 7 diffe en i eous humou s, wi h 5 le els o blood
con amina ion o each one. Finally, we pe o med a mic oscopic con ol o haema ic
con amina ion o he diffe en aliquo s by Pap s aining a e concen a- ion by
ul acen i uga ion.
3. Resul s and discussion
3.1. Valida ion
Linea i y was e ified o all he compounds excep UA, whe e he esul s we e no su ficien ly
sa is ac o y o de e mine i s coe ficien ( 2) and esiduals. The calib a ion cu es o he es o
he compounds we e fi ed wi h 1/x2-weigh ed ac o , and he 2 we e abo e 0.99. The esiduals
we e wi hin 20% a LOQs and 15% a o he calib a o concen a ions o all compounds. The
LOD anged be ween 0.25 and 2.5 mM, and he LOQ was he lowes poin o he calib a ion
ange. These esul s a e summa ized in Table 2. The LOD and LOQ alues achie ed wi h his
me hod a e app op ia e o Hx de e mina ion, gi en ha he alues o his analy e ound in
p e iously pape s anged be ween 10.67 mM o 125.27 mM in 176 cases analyzed by Muñoz e
al. [9] and be ween 4.8 mM and 45.6 mM in 120 cases analyzed by Jashnani e al. [7].
Table 3 shows he esul s o imp ecision and analy ical eco e y. These we e sa is ac o y o all
es ed compounds excep UA, which had p oblems wi h linea i y. Table 4 shows ex ac ion
e ficiency, p ocess e ficiency and ma ix effec esul s o Hx, G, X, UA and IS. Ex ac ion e ficiency
anged om 62.6% o 114.4%, wi h alues highe han 50% o all analy es. Hx, G and IS showed
ion enhancemen anging om 14.1% o 137.1%, and UA ion supp ession. X showed no
significan ma ix effec . In all cases % CV was below 20%. P ocess e ficiency anged om 25.3%
om 127.6%.
Good quan ifica ion o eal specimens wi h non-ma ix calib a ion cu es was achie ed o Hx, G
and X. Adequa e accu acy was ound, wi h alues o %MRE below 15% (-0.3% o Hx, -12.8 o G
and -3.3 o X). P ecision esul s (%CV was 7.7 o Hx, 12.7 o G and 17.4 o X) we e sa is ac o y
o Hx and G, wi h alues below 15%, and sligh ly highe o X, bu below 20%.
Ve y ew p e ious pape s epo [Hx] de e mina ion by LC– MSMS [22,23], and hese cases used
u ine samples. No p e ious me hods ha e been de eloped o de e mina ion o his analy e in
i eous humou , despi e he high sensi i i y and specifici y o his echnique.
3.2. E alua ion effec o blood con amina ion
Mac oscopically, he samples showed no colo a ion al hough he i eous humou s we e
con amina ed wi h blood. Mic oscopically, a e s aining he aliquo s, he e was no e idence o
e y h ocy es. These samples seem o show a dec ease in Hx concen a ions, as well as in G and
X concen a ions, in ela ion o inc eased blood concen a ions. Howe e , no significan
co ela ion be ween he 5 le els o blood con amina ion was obse ed in any o he 15 samples
analyzed. Table 5 shows he alues ob ained o he selec ed analyzed samples o i eous
humou and hei le el o blood con amina ion.
Applying he o mula p oposed [12] o de e mining PMI, and gi en he alue o [Hx], he change
in he cen al alue o PMI o he confidence in e al es ima ed can cause a a ia ion in a PMI
es ima e om 14.4 h o an uncon amina ed sample o 8.6 h o a con amina ed one (see Table
5, Specimen 1. The mean a ia ion o o al specimens was 4.02 h). No s a is ically significan
ela ion- ship be ween he inc ease in blood concen a ion and changes in he es ima e we e
ound, which u he demons a es he impossibili y o es ima ing PMI by means o de e mining
blood Hx because an e oneous PMI alue would be ob ained om con amina ed samples. Such
modifica ions also affec G and X (Table 5).
Howe e , UA showed cha ac e is ic beha iou and all samples wi h blood con amina ion showed
a o al disappea ance o he wo MRM ansi ions o his analy e. Al hough UA alida ion was
no sa is ac o y, in e ms o linea i y, analy ical eco e y and p ecision, i was possible o make a
quali a i e de e mina ion. Fig. 1 p esen s he ch oma og am o a eal specimen o i eous
humou be o e and a e blood con amina ion wi h 1 mL o blood dilu ion in saline solu ion a
5%. A o al disappea ance o UA was clea ly obse ed.
This me hod makes i possible o iden i y hose samples whe e blood con amina ion is due o
imp ope ex ac ion o he i eous humou . E en a low le els o con amina ion (when no
mic oscopically e iden ), and be o e Hx concen a ions a e affec ed, he wo MRM ansi ions o
UA disappea , hus enabling he samples o be disca ded, in he same manne as he clea ly
con amina ed samples, and hus a oid e oneous esul s in PMI [2,4,9,18]. The p ocedu e has
been applied in he ou ine analysis o hypoxan hine in i eous humou . A o al o 186 clea
i eous, ob ained by well- ained o ensic pa hologis wi h long expe ience in ob aining he
samples, we e analyzed. O hese, 7 showed o al disappea ance o he wo ansi ions o u ic
acid al hough no mac oscopically blood con amina ion was obse ed, p o ing he ele ance o
using his new analy ical p ocedu e.
4. Conclusions
A new LC–MSMS me hod was de eloped o quan i a i e and quali a i e analysis o Hx, G and X,
and quali a i e de e mina ion o UA. The me hod was ully alida ed in e ms o linea i y,
sensi i i y, imp ecision, analy ical eco e y, ex ac ion and p ocess e ficiency, and ma ix effec .
Du ing he p ocess o i eous humou ex ac ion blood con amina ion can occu as a esul o
ascula inju y and such con amina ion could dis o he esul s o PMI. Howe e , by moni o ing
UA i has been possible o es ablish a new c i e ion o iden i y specimens o i eous humou
wi h blood con amina ion, e en when no colo a ion is e iden . Fo hese easons i is a me hod
o choice o he es ima ion o he PMI by means o he de e mina ion o Hx and whe e haema ic
con amina ion can be objec i ely demons a ed.
Acknowledgmen s
We g a e ully acknowledge he suppo by INCITE (Conselle ía de Inno ación e Indus ia, Xun a
de Galicia), P ojec Numbe INCITE09 228 166 PR.
P esen ed in pa a he 19 h Mee ing o he In e na ional Associa ion o Fo ensic Sciences,
Funchal.
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