*Co esponding au ho : K ishnaphanis i Ponnekan i.
Copy igh © 2025 Au ho (s) e ain he copy igh o his a icle. This a icle is published unde he e ms o he C ea i e Commons A ibu ion Liscense 4.0.
A e iew: On HPLC me hod o es ima ion o me hylcobalamin
K ishnaphanis i Ponnekan i *, Addanki Anusha, Gee ha Jummidi, Sai Hala a hu and Shi a eddy Ka na i
Depa men o Pha maceu ical Analysis, Malla Reddy Ins i u e o Pha maceu ical Sciences, Maisammaguda, Kompally.
India.
Wo ld Jou nal o Biology Pha macy and Heal h Sciences, 2025, 21(01), 282-289
Publica ion his o y: Recei ed on 30 No embe 2024; e ised on 08 Janua y 2025; accep ed on 10 Janua y 2025
A icle DOI: h ps://doi.o g/10.30574/wjbphs.2025.21.1.0018
Abs ac
Me hylcobalamin, a i al i amin B12 analog, is suscep ible o deg ada ion unde he mal s ess, leading o changes in
PH. T igeminal neu algia, diabe ic neu opa hy, acial pa alysis and megaloblas ic anemia can be ea ed by he use o
Me hylcobalamin injec ion. A new simple, p ecise, economically iable and e icien High-Pe o mance Liquid
Ch oma og aphic (HPLC) me hod o he es ima ion o Me hylcobalamin (MeB12) in bulk d ugs and pha maceu ical
dosage o mula ions was de eloped and alida ed. The me hod was alida ed o linea i y, accu acy, p ecision, LOD,
LOQ and obus ness as pe ICH guidelines. The me hod was es ed wi h comme cially a ailable MeB12 d ugs and he
concen a ion me hylcobalamine was ound o be accu a e.
Keywo ds: Me hyl cobalamin; HPLC; Me hod alida ion; Vi amin
1. In oduc ion
Chemically Me hylcobalamine (Me B12 o Me Cbl, MF: C63H91CoN13O14P) belongs o cobalamin g oup compounds [1].
Me hylcobalamin had p onounced suscep ibili y o hyd olysis unde acidic, alkaline, and pho oly ic condi ions.
Me hylcobalamin ollowed pseudo- i s -o de kine ics upon exposu e o acidic and alkaline hyd olysis wi h highes
s abili y a pH 5 and leas s abili y a pH 2 [2]. Me hylcobalamin is used o he ea men o megaloblas ic anaemia.
Me hylcobalamine (MC; ca banide; cobal ; [5-(5,6-dime hylbenzimidazol-1 p opanoylamino] p opan-2-yl hyd ogen
phospha e) is de i a i e o m o i amin B12. I is used o he ea men o pe iphe al neu opa hy, diabe ic neu opa hy
[3]. Me hylcobalamin is essen ial o cell g ow h and eplica ion. In he p esen in es iga ion an a emp was made o
de elop an HPLC me hod. Me hylcobalamin is he me abolically ac i e o m p e equisi e o cobalamin-dependen
enzyme.
1.1. Me hylcobalamin
Me hylcobalamin Li e a u e e iew e ealed ha i amin B12 is de e mined adi ionally by mic obiological assay using
Lac obacillus; al hough he assay is sensi i e, i is no speci ic as inac i e cobalamin can in e e e wi h mic oo ganism.
The mos scien i ic and en i onmen al majo signi icancan ole o MeB12 is biome hyla ion o ce ain hea y me als I is
used in pe iphe al neu opa hy, diabe ic neu opa hy and o p elimina y ea men o amyo ophic la e al scle osis.
S uc u ally, cobalamins ha e cen ally coo dina ed cobal a om whose i h posi ion is co alen ly occupied by he
g oups such as CH3. Fo he di e en comme cial d ugs o me hylcobalamin namely Neu okind, Neu os a , Keeneu on
and Me ck sample we e used o es he accu acy o he de eloped me hods. Me hylcobalamine is o icial in USP, EP and
BP. Me hylcobalamine is a ou ine es ima ion in li e , plasma, milk, in es inal luids and aeces has been epo ed.
Me hylcobalamine is essen ial o cell g ow h and eplica ion.
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Figu e 1 Chemical S uc u e o Me hyl cobalamin
Figu e 2 Mechanism o ac ion o me hylcobalin
Me hylcobalamine is essen ial o cell g ow h and eplica ion. Me hylcobalamine a e na u al o ms o i amin B12
comme cially a ailable in di e en ypes o pha maceu ical dosage o ms. Me hylcobalamine is he me abolically ac i e
o m p e equisi e o cobalamin- dependen enzyme unc ion o he d ug. I ex ensi e deg ada ion occu s, limi
exposu e should be conside ed [6].
2. High pe o mance liquid ch oma og aphy
High-pe o mance liquid ch oma og aphy (HPLC) s ands impu i ies, syn hesis in e media es, and deg adan s. As a as
a powe ul analy ical ool in mode n chemis y. HPLC is an analy ical echnique in which solu es a e esol ed by
di e en ial a es o elu ion as hey pass h ough a ch oma og aphic column [6]. Now a day e e sed-phase
ch oma og aphy is he mos commonly used sepa a ion echnique in HPLC due o i s b oad applica ion ange. I is
es ima ed ha o e 65% (possibly up o 90%) o all HPLC sepa a ions a e ca ied ou in he e e sed phase mode [7].
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HPLC is quan i a i e and quali a i e d ug p oduc analysis, playing p ized o i s p ecision in bo h quan i a i e and a
pi o al ole in de e mining d ug p oduc s abili y [8].
Figu e 3 Ins umen a ion o HPLC
3. Types o HPLC
Depending upon subs a e used i.e. s a iona y phase used, he HPLC was di ided in o ollowing ypes:
3.1. Re e se Phase HPLC
Re e sed phase ch oma og aphy has ound bo h analy ical and p epa a i e applica ions in he a ea o biochemical
sepa a ion and pu i ica ion. Uses wa e -o ganic as mobile phase, columns may be C18 (ODS), C8, phenyl, T ime hyl Silane
(TMS), cyano as a s a iona y phase [8].
3.2. No mal Phase HPLC
I is i s choice o mix u es o isome s and o p epa a i e scale HPLC and second choice o lipophilic samples ha
canno dissol e well in wa e -o ganic mix u es [8].
3.2.1. Bio-a ini y ch oma og aphy
Based on speci ic in e ac ions, making a ini y ch oma og aphy a aluable echnique in biochemis y and p o ein. mos
samples especially neu al o non-ionized compounds, ha dissol e in wa e o ganic mix u es [9].
3.3. RP-HPLC (Re e sed-Phase HPLC)
RP-HPLC is widely used o i s abili y o sepa a e compounds based on hyd ophobici y, p o iding e ec i e analysis in
a ious ields, including pha maceu icals, biochemis y [9].
3.4. Ins umen a ion
The HPLC ins umen a ion in ol es he ollowing pa s:
3.4.1. Da a collec ing de ice (compu e ):
The compu e no only con ols all he modules o he HPLC ins umen bu i akes he signal om he de ec o and
uses i o de e mine he ime o elu ion ( e en ion ime) o he sample componen s (quali a i e analysis) and he amoun
o sample (quan i a i e analysis) [10].
3.4.2. Sample Injec ion:
The injec o can be a single injec ion o an au oma ed injec ion sys em. Re e ing o load / injec al e in s ep (2e) e i y
al e handle is in he load posi ion. And low a e is 3ml/min. I al e handle is no in load posi ion he plug will be
o ced ou upon elease by 2000 PSI. This should s a he da a acquisi ion. The display should change om “Wai ing
o Injec ion” o Running” [10].
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3.4.3. Clean Sy inge:
Rinse sy inge ho oughly wi h DI wa e un il clean [11].
3.4.4. Pumps:
I is necessa y o pump he eluen a a cons an low a e and p essu e. Con en ional, analy ical HPLC pumps a e he
mos common ype, bu semi-mic o and a p epa a i e A pump can be compa ed o he human hea which con inuously
pumps blood h oughou he body bu h ough he human hea can wi hs and changes in blood p essu e wi hin a
speci ied limi due o s ess and s ain he HPLC pump is equi ed o deli e a low o mobile phase a cons an p essu e
and low a e. e pumps a e also used depending on he ange o he eluen low a e equi ed [11].
3.4.5. S a iona y Phase:
The s a iona y phase in HPLC e e s o he solid suppo con ained wi hin he column o e which he mobile phase
con inuously lows. The chemical in e ac ions o he s a iona y phase and he sample wi h he mobile phase, de e mines
he deg ee o mig a ion and sepa a ion o he componen s con ained in he sample. Columns con aining a ious ypes
o s a iona y phases a e comme cially a ailable.
3.4.6. Columns
Columns a e usually made o polished s ainless s eel, a e be ween 50 and 300 mm long, and ha e an in e nal diame e
o be ween 2 and 5 mm. The column is one o he mos impo an componen s o he HPLC ch oma og aph because he
sepa a ion o he sample componen s is achie ed when hose componen s pass h ough he column.
Column empe a u e con ol: Fo some applica ions, close con ol o column empe a u e is no necessa y and columns
a e ope a ed a oom empe a u e.
Table 1 Columns No mal s Re e sed Phase
No mal
Re e se
Packing pola i y
High
Low
Sol en pola i y
Low
High
Elu ion o de
Non-pola i s , hen pola
Pola i s , hen non-pola
E ec o inc easing sol en pola i y
Dec eases e en ion ime
Inc eases e en ion ime
3.4.7. De ec o
The componen s elu ed om he column a e de ec ed, and he de ec ion da a a e con e ed in o an elec ical signal. The
de ec o is selec ed o sui he sample. The de ec o p o ides an ou pu o a eco de o compu e ha esul s in he
liquid ch oma og am
Table 2 Majo ypes o de ec o s
UV de ec o
The ligh sou ce is a D2 lamp. This de ec o is used mainly o de ec componen s ha ing an
abso p ion wa eleng h o 400 nm o less in he ul a iole egion.
UV-VIS de ec o
A D2 lamp and a W lamp a e used as he ligh sou ce. This de ec o is e ec i e in he de ec ion
o colo ing componen s such as dyes and s ains because o co e age o he isible ligh egion
Diode a ay
de ec o (DAD)
Da a on he spec um om he ul a iole o isible ligh ange is also collec ed
Fluo escence (FL)
de ec o
Fluo escen subs ances can be de ec ed speci ically wi h high sensi i i y.
Mass spec oscopy de ec o : Uni e sal de ec o The MS de ec o can some imes iden i y he compound di ec ly since
i s mass spec um is like a inge p in and is unique o ha compound.
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3.4.8. Ch oma og aph Da a Acquisi ion
sa e ch oma og am, close ch oma og am - go o Acquisi ion – Quick S a – Selec Me hod – Be su e o selec he me hod
p e iously used, make any no es conce ning injec ion olume e c. I you ge an “In use” e o close p og am and eboo
compu e .
3.4.9. Size Exclusion Ch oma og aphy (SEC)
This echnique is widely used o he molecula weigh de e mina ion o polysaccha ides. In SEC, he e is no in e ac ion
be ween he sample compounds and he column packing ma e ial. I is also use ul o de e mining he e ia y s uc u e
and qua e na y s uc u e o pu i ied p o eins [11].
3.5. Me hod de elopmen o HPLC
The goal o he HPLC-me hod is o y & sepa a e, quan i y he main ac i e d ug, any eac ion impu i ies, all a ailable
syn he ic in e -media es and deg ada ion.
Figu e 4 Flow cha o me hod de elopmen o HPLC
Ch oma og aphic in o ma ion. The PC coo dina es he eac ion o he indica o o e e y pa and places i in o a
Ch oma og aph ha is any hing bu di icul o in e p e .
3.6. Componen s o me hod alida ion
• Accu acy
• P ecision
• Linea i y
• De ec ion limi
• Quan i a ion limi
• Speci ici y Range
• Robus ness
• Ruggedness
3.6.1. Accu acy
To s udy o he eliabili y sui abili y and accu acy o he me hod eco e y expe imen s a e ca ied ou o MeB12 in
wo s ages and he a e age eco e y o d ugs was calcula ed. The accu acy o he p oposed me hod was assessed by
iplica e analysis o h ee di e en concen a ions (40, 80, and 120 μg/ml).
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he assay was pe o med as pe he es me hod. Then om hese esul s he alues calcula ed we e, pe cen age
eco e y and quan i y p esen (mcg).
3.6.2. P ecision
P ecision was e alua ed a he epea abili y and in e media e p ecision le els. Fo epea abili y analysis, six
independen po ions o a sample solu ion o Me hylcobalamin. The in a- and in e day p ecision was ca ied ou by
analyzing h ee concen a ions using in he same day o h ee successi e days. %RSD o he esul s ob ained. The alues
o %RSD wi hin a day, day o day a ia ion (<1%) p o es ha he me hod is p ecise.
3.6.3. Linea i y
The linea i y o measu emen was e alua ed by analyzing di e en concen a ions o he s anda d solu ions MeB12. A
calib a ion cu e was ob ained o MeCbl by iplica e injec ion o MeCbl s anda d in he ange o 2–160 μg/ml. Mean
peak a ea o each concen a ion was plo ed agains he co esponding concen a ion linea i y o s anda d
Me hylcobalamine powde was de e mined wi h he help o UV spec opho ome e a 353 nm and eco ded hei
abso bance.
3.6.4. De ec ion and quan i a ion limi s
Limi o de ec ion (LOD) and limi o quan i a ion (LOQ) we e calcula ed using he ollowing equa ions: LOD = 3.3* σ/S,
LOQ = 10* σ/S, espec i ely, whe e S is he alue o he calcula ed slope and σ is he esidual s anda d de ia ion o
eg ession line, and LOD and LOQ we e ound o be 0.6556 and 1.9867 μg/ml, espec i ely.
LOD = 3.3 × S /SD and LOQ = 10 × S /SD
3.6.5. Speci ici y
Speci ici y was de e mined by quan i ying MeCbl in he p esence o p obable excipien s in di e en ypes o dosage
o ms ( able , ampoule and o al dissol able ilm) also in p esence o all po en ial deg ada ion p oduc s p oduced unde
s ess deg ada ion condi ions. The o ced deg ada ion s udy o he iple combina ion d ug e ealed ha d ug was
deg aded unde he in luence o acid, alkali, he mal, wa e , hyd ogen pe oxide and pho oly ic condi ions.
3.6.6. Ruggedness
In e -day a ia ions we e pe o med by using six eplica e injec ions o s anda d and sample solu ions o
concen a ions which we e p epa ed and analyzed by di e en analys on h ee di e en days o e a pe iod o one
week. Ruggedness also exp essed in e ms o pe cen age ela i e s anda d de ia ion [11].
3.6.7. Robus ness
The analy ical me hod obus ness was es ed by e alua ing he in luence o mino modi ica ions in HPLC condi ions on
sys em sui abili y pa ame e s o he p oposed me hod such as he composi ion o he mobile phase, empe a u e and
low a e. ha he pe cen o eco e y was wi hin accep able limi s and he %RSD was wi hin limi o no mo e han
2.0%. The ailing ac o s and numbe o heo e ical pla es we e ound wi hin accep able limi s as well.
3.6.8. Range
The ange o me hod is in e al be ween he uppe lowe le els o an analy e ha ha e been de e mined
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Table 3 Li e a u e e iew o Me hyl cobalamin
S.NO
COLUMN
MOBILE PHASE
RESULT ss
REFEERENCE
1
K omasil C18
Me hanol: wa e ( a io 20:80 / )
Re en ion ime: 6 min
Linea i y: 0.999
Accu acy: 99.53%
P ecision:0.632-0.764%
LOD: 0.15 µg/ml
LOQ: 0.5 µg/ml
[12]
2
USP C18
Me hanol: ACN (55:35:10, / )
Re en ion ime: 6 min
Linea i y: 2–160 μg/ml
Accu acy: 96.17%
P ecision: 96.17 %
LOD: 0.6556 μg/ml
LOQ: 1.9867 μg/ml
[13]
3
Cosmosil C18
Me hylcobalamin: dis illed wa e (50μg/ml)
Re en ion ime: 6 min
Linea i y: 10-50 μg/ml
Accu acy: 99.29 -100.5 %
LOD: 0.00342 μg/ml
RSD: 1.33
[14]
4. Conclusion
A s abili y indica ing assay me hod o MeCbl de e mina ion was de eloped and alida ed. The p oposed me hod is
simple, accu a e and applicable o he analysis o MeB12 in bulk d ugs and able o mula ions. Pe cen age eco e y was
p o ed o be in pa wi h ICH guidelines and he p oposed me hod was accu a e and simple.
This me hod was alida ion o linea i y, accu acy, p ecision, epea abili y, LOD and LOQ o Me hylcobalamin.
Compliance wi h e hical s anda ds
Disclosu e o con lic o in e es
No con lic o in e es o be disclosed.
Re e ence
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