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Ad ances in Analy ical Me hods o Py idoxine
Hyd ochlo ide and Combina ion The apies: A
Comp ehensi e Su ey
1*Judy Jays. 2P adan Jain,
1Associa e P o esso , 2MPha m s uden ,
1Depa men o Pha maceu ical Chemis y
1Facul y o pha macy, M S Ramaiah Uni e si y o Applied sciences, Bengalu u, India
1judyjays.py.ph@ms uas.ac.in, 2 p [email protected]
Abs ac
Py idoxine Hyd ochlo ide plays a c ucial ole in nume ous physiological p ocesses, and i s de iciency can lead o a ious
heal h complica ions. The de elopmen o p ecise analy ical echniques is essen ial o ensu ing he quali y, sa e y, and e icacy
o Py idoxine Hyd ochlo ide o mula ions, bo h as s andalone supplemen s and as componen s o combina ion he apies. This
s udy p esen s a comp ehensi e o e iew o cu en ad ancemen s in analy ical me hodologies used o he measu emen and
e alua ion o py idoxine hyd ochlo ide and i s combina ion he apy. Unde s anding he shi ing landscape o analy ical
me hods is becoming mo e essen ial as he impo ance o py idoxine hyd ochlo ide expands om solo o mula ions o
complica ed combina ion medicines. The u iliza ion o a ious analy ical me hods, such as ch oma og aphic condi ions o
me hod de elopmen and alida ion pa ame e s in ensu ing he quali y, sa e y, and e icacy o hese pha maceu ical
o mula ions a e e iewed. Fu he mo e, he challenges, u u e p ospec s, and po en ial esea ch di ec ions in he ield o
analy ical chemis y o Py idoxine Hyd ochlo ide and combina ion he apies a e discussed.
Key wo ds: HPLC, RP-HPLC, Valida ion, Py idoxine Hyd ochlo ide, Me hod De elopmen .
INTRODUCTION
Vi amin B, a g oup o wa e -soluble i amins, is essen ial o a ious physiological unc ions wi hin he human body, including ene gy
me abolism, ne ous sys em unc ion, and ed blood cell o ma ion. Among he a ious B i amins, i amin B6, also known as
py idoxine, s ands ou o i s di e se oles in main aining heal h and well-being. The h ee di e en o ms o Vi amin B6 a e
Py idoxine, Py idoxal and Py idoxamine in which py idoxine is ound in na u al sou ces. S uc u e o hese h ee o ms depend upon
he subs i uen a ou h posi ion o he py idine molecule [1]. Body ans o ms all h ee de i a i es o i amin B6 in o py idoxal
phospha e. The p ima y physiologically ac i e o m o py idoxine o i amin B6, is py idoxal 5-phospha e [2]. A co- ac o in a numbe
o enzyma ic p ocesses ela ed o amino acid me abolism is py idoxine. In o de o ca y ou he a ious me abolic ans o ma ions o
amino acids he py idoxal phospha e o m is essen ial. Mo eo e , i plays a ole in he gene a ion o neu o ansmi e s like
no epineph ine and se o onin and he ans o ma ion o s o ed glycogen in o glucose o ene gy. Comme cially accessible ee
i ame s o B6 a e c ys alline hyd ochlo ides such as py idoxine hyd ochlo ide and py idoxamine dihyd ochlo ide. Because o i s
supe io s abili y han Py idoxal and Py idoxamine, py idoxine hyd ochlo ide is used as s anda d and he sole o m u ilized in ood
o i ica ion and pha maceu ical o mula ions [3]. Py idoxine is well acknowledged o be i al in human nu i ion. I has been
es ablished ha a pa ien s will be de icien o i amin B6 i de iciencies in o he g oup o he B i amin amily a e ound. As a esul ,
py idoxine he apy can be combined wi h o he i amins’ he apy. Howe e , cu aneous and cen al ne ous sys em abno mali ies a e
no ably ela ed wi h py idoxine insu iciency.
Py idoxine is ound in a a ie y o oods such as poul y, nu s, mea , ish, whole g ains and o i ied ce eals. I is also a ailable as a
nu i ional supplemen in he o m o py idoxine hyd ochlo ide. Daily in ake o i amin B6 will change based on a numbe o a iables,
including age, sex, and s age o li e. Vi amin B6 is equen ly inco po a ed in combina ion medica ions. Heal hca e p o essionals hope
o add ess a ious aspec s o he p oblem by combining py idoxine hyd ochlo ide wi h o he medica ions, esul ing in a mo e
comp ehensi e and success ul ea men plan [4,5].
Analy ical me hods a e undamen al ools in scien i ic esea ch, indus y, and a ious ields o applied science. These me hods
encompass a wide ange o echniques designed o iden i y, quan i y, and cha ac e ize subs ances o componen s wi hin a sample.
F om pha maceu ical de elopmen o en i onmen al moni o ing, analy ical me hods play a c ucial ole in ensu ing p oduc quali y,
sa e y, and compliance wi h egula o y s anda ds. The choice o analy ical me hod depends on a ious ac o s, including he na u e o
he sample, he analy e o in e es , he equi ed sensi i i y and selec i i y, and he desi ed le el o p ecision and accu acy. Analy ical
me hods can be b oadly ca ego ized in o quali a i e and quan i a i e echniques. Quali a i e me hods a e used o iden i y he p esence
o absence o speci ic subs ances o componen s, while quan i a i e me hods p o ide in o ma ion abou he concen a ion o amoun
o a pa icula analy e in a sample. Among he ple ho a o analy ical echniques a ailable, e e sed-phase high-pe o mance liquid
ch oma og aphy (RP-HPLC) s ands ou as one o he mos e sa ile and widely u ilized me hods o he sepa a ion, iden i ica ion, and
quan i ica ion o compounds in complex mix u es [6].
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Table 1 D ug P o ile
Name
Py idoxine Hyd ochlo ide
Molecula o mula
C8H11NO3
Molecula weigh
169.18g
IUPAC name
4,5 - bis(hyd oxyme hyl)-2-me hylpy idin -3-ol
Solubili y
Soluble in Wa e , DMSO, me hanol, e hanol and
ace one
Mel ing poin
162°C
S uc u e
METHODOLOGY
The ollowing keywo ds we e used in a li e a u e sea ch ac oss se e al da abase sou ces, including PubMed and Google Schola :
HPLC, RP-HPLC, Valida ion, and Py idoxine Hyd ochlo ide. By using he sui able il e , he sea ch was ailo ed o ind he a icles
ha we e mos ele an o his e iew.
ANALYTICAL METHODS
Tad i Shabnam bi Ga oo Kha, e .al in 2023 de eloped a Re e se phase-HPLC me hods o es ima ing simul aneously Mela onin
and Py idoxine in pha maceu ical o mula ion. Agilen wi h an au o sample G adien Sys em DAD (Diode A ay De ec o ) wi h a
C18 column (250 mm X 4.6 mm) wi h a pa icle size o 5μm used o he RP-HPLC p ocess. The mobile phase was ace oni ile
40%:60% wa e wi h 0.1% TEA (PH 6.2 wi h OPA) a a low a e o 1.0 mL/min and he de ec ing wa eleng h was 265 nm. Mela onin
and py idoxine we e shown o ha e e en ion o 2.008 and 3.042 min, acco dingly. ICH guidelines we e ollowed in he alida ion o
he de eloped app oach. The ICH guidelines limi a ions we e me by he linea i y, accu acy, ange, and obus ness. The app oach was
he e o e de e mined o be s aigh o wa d, accu a e, p ecise, a o dable, and epea able. [7]
A. Cha an, e .al in 2023 de eloped a RP-HPLC echnique o es ima e and alida e impu i y analysis o py idoxine in a la ge
quan i ies and o mula ion. Quali y by design (QbD) s a egy was used o ine- une he me hod, assu ing i s du abili y and
dependabili y. RP-HPLC me hod was ca ied ou using mobile phase composed o ace oni ile: wa e (90:10 / ) a a low a e o
1mL/min and column wi h dimension (4.6 x 250mm, 5µm) was employed o he ch oma og aphic sepa a ion which was main ained
a 30°C. UV (DAD) de ec o was used o measu e he abso bance o e luen a 281nm. The e en ion pe iod o py idoxine was 6.3
min and he alida ion pa ame e s such as accu acy, linea i y and p ecision we e e alua ed a en i ely as c i ical pe o mance
measu es. The me hod exhibi ed ema kable p ecision wi h a ela i e s anda d de ia ion (RSD) o less han 2%, g ea eco e y a es
(100.2 and 101.2%), and a obus co ela ion coe icien o 0.999. The me hod can be employed o impu i y p o iling and can be
employed o p olonged s abili y es ing as i shows s abili y. [8]
D . A.Y. Ghodke, e .al in 2023 o he pu pose o quan i a i ely de e mining me hyl cobalamin, alpha lipoic acid, py idoxine HCl
and olic acid in bulk and comme cial dosages, an accu a e, ep oducible and speci ic Re e se phase-HPLC me hod was p oposed
and he me hod was alida ed by using di e en pa ame e s as pe ICH guidelines. The me hod was pe o med by using column
Agilen Zo bax Bonus RP (250 mm x 4.6 mm) wi h 5µm pa icle size. Mobile phase con aining 0.1% O ho-phospho ic acid and
me hanol in he a io o 50:50 was used a a low a e o 0.8ml/min o e icien sepa a ion and de ec ed a 270nm. Re en ion du a ion
was es ima ed o be 6.0 minu es wi h 10–30µg/mL linea i y ange, 0.9980 was he co ela ion coe icien and he eco e y a e was
ound o be 99.8% acco dingly. [9]
A. El-Hadi, e .al in 2022 de eloped RP-HPLC me hod con aining diode a ay de ec o (DAD) o measu e doxylamine succina e
(DOX) and py idoxine HCl (PYR) in he exis ence o DOX oxida ion deg ada ion p oduc (DOX DEG). The analysis was pe o med
on X e a C18 column wi h 100mm x 4.6 mm dimensions using 0.01M phospha e bu e and e hanol in he a io o 90:10 / a pH 5
and lowed a a a e 1mL/min wi h 10min un ime. Abso bance was measu ed a 254nm by using DAD. Py idoxine HCl, doxylamine
succina e and DOX-DEG we e ound o ha e e en ion ime o 1.41min, 5.59min and 7.36 min, acco dingly. Valida ion was done
using di e en pa ame e s ollowing ICH guidance, he linea i y ound in he le el o 10-120µg/mL (PYR) and 5-100µg/mL (DOX)
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wi h co ela ion o 0.9997 and 0.9999, espec i ely and exhibi ed excellen eco e y a es (100.41% and 100.04%). Hence, he
de eloped me hod could be p ac ical and sa e subs i u e o ou ine examina ion o he d ugs unde s udy. [10]
Ma ique Elizabe h Aucamp, e .al in 2021 The mos e ec i e, simul aneously sepa a ing and iden i ying o Te izidone (TZD) and
Py idoxine (PDX) we e accomplished employing an isoc a ic sol en sys em ha included ul apu e wa e and ace oni ile (30:70%
/ ) wi h 1 mL glacial ace ic acid. A 5 μm column measu ing 150 x 6 mm used, wi h a 260 nm de ec ion wa eleng h. The me hod's
linea i y, accu acy, p ecision, obus ness, speci ici y, limi o de ec ion (LOD), limi o quan i ica ion (LOQ) and s abili y o he
solu ion we e all alida ed and alida ion demons a ed ha his app oach was app op ia e and dependable o he accu a e
simul aneous de ec ion and measu emen o TZD and PDX. Pe cen age eco e y ob ained o TZD and PDX is 98%. [11]
Saini, e .al in 2021 de eloped a Re e se phase– HPLC echnique o alida ing and es ima ing Me hionine, Nico inamide and
Py idoxine hyd ochlo ide simul aneously in combina ion dosage o m. Me hanol and 0.01M sodium ace a e we e used as a mobile
phase in he a io o 600:400( / ) a a pH 5.2 and a low a e o 1mL/min. Ine sil-ODS (Oc adecyl Silica) C18 column (250mm ×
4.6mm) wi h 5µm pa icle size was used o sepa a ion which was main ained a 25°C and he eluen was measu ed a 247nm.
Me hionine, Py idoxine hyd ochlo ide and Nico inamide we e ound o ha e e en ion ime o 1.4min, 2.2min and 4.4 min
espec i ely, whe e he co ela ion coe icien was 0.999 wi h concen a ion o 10-30µg/mL. Reco e y s udies we e conduc ed in
o de o assess he accu acy. Me hionine, py idoxine hyd ochlo ide, and nico inamide we e ound o ha e eco e y a es o 99.9%,
98.95%, and 99.8%. [12]
Pa el, e .al in 2021 epo ed ha use o de i a izing agen s imp o es he ch oma og aphic beha iou . Leucine was con e ed in o
Fmoc-Leucine de i a i e as a de i a izing agen in o de o educe ola ili y and imp o e ch oma og aphic beha iou o an analy e.
Fo he de e mina ion o Fmoc-Leucine and Py idoxine HCl simul aneously, RP-HPLC me hod and UV- isible spec oscopic me hod
we e de eloped using sol en sys em composed o 0.1% OPA wi h ACN: wa e (50:50% / ) a a low a e o 1mL/min. The column
used was Zo bex C18 (250mm × 4.6mm) and main ained a 40°C, bu he analysis was accomplished a ambien empe a u e o 25
°C and he de ec ion o e luen was done a 285nm. The e en ion pe iod o Py idoxine HCl as well as Fmoc-Leucine ob ained a 3.4
and 14.3 minu es wi h a un ime o 10min. The me hods we e alida ed using se e al pa ame e s like accu acy, p ecision, linea i y,
LOD and LOQ, obus ness acco ding o ICH guidelines. Py idoxine hyd ochlo ide and Fmoc-Leucine we e shown o ha e a
co ela ion coe icien o 0.999:0.997 and 0.999:0.995 and wi h pe cen age eco e y o 98.6%, 98.7%, 99.2%, 99.3% acco dingly.
[13]
Bachmann, e .al in 2020 o he pu pose o quan i ying each B6 i amins such as py idoxamine (PM), py idoxine (PN), py idoxal
(PL), py idoxamine phospha e (PMP) and py idoxine glycoside (PNG) in g ains, ui s and ege ables LC-MS/MS and s able iso ope
dilu ion assay (SIDA) was de eloped. The R o each B-g oup i amins was ound o be 1.9min o PN, 1.03min o PM, 1.57min o
PL, 1.06min o PMP and 2.12 min o PNG. Valida ion was done by using s a ch ma ices and o each analy e, he limi s o
quan i ica ion and de ec ion we e 0.0085 o 0.059 mg/kg; 0.0028 o 0.02 mg/kg, acco dingly. The pe cen age eco e ies anged om
92-111%. Addi ionally, using LC-MS/MS and SIDA, he i s quan i ica ion o PNG was ca ied ou wi hou he need o enzyma ic
p ocesses o inconsis en in e nal s anda ds. [14]
T. Benjamin, e .al in 2019 A no el isoc a ic e e se phase- HPLC echnique was iden i ied o he quan i ica ion o isoniazid and
py idoxine able s simul aneously. The mo's Hype sil BDS column (250 x 4.6 mm, 5μ) was used o pe o m he sepa a ion. The mobile
phase was composed o ace oni ile and phospha e bu e (pH 4) in a 75:25 / a io. Du ing he 6-minu e un ime, injec ion olume
was 10 μL, column o en empe a u e was 30°C, low a e was 1.0 mL/min, and a pho odiode a ay de ec o (PDA) was used o
de ec ion a 267 nm. Isoniazid and py idoxine we e ound o ha e e en ion imes o 3.5 and 4.3 minu es espec i ely, which sugges s
a compa a i ely sho e analysis ime. The p ocedu e was e i ied in acco dance wi h ICH s anda ds. The s udy p o ides a no el,
p ecise, epea able, and eliable echnique o he simul aneous es ima ions o he wo d ugs. [15]
H. Hashem, e .al in 2018 de eloped RP-HPLC me hod by u ilizing QbD s a egy o he es ima ing Le e i ace am and Py idoxine
HCl in able dosages. The d ugs we e gi en oge he o p e en he py idoxine HCl de ici which was b ough on by an iepilep ic
le e i ace am. BDS Hype sil C8 column o (250mm × 4.6mm) dimensions was employed o he sepa a ion. Me hanol and bu e
(KH2PO4) we e used as isoc a ic mobile phase in he a io o 38.4: 61.6( / ) wi h pH 3 and 0.8mL/min a e o low. De ec ion o he
e luen was done a 214nm. Valida ion was done as pe ICH guidelines, he linea i y ange o le e i ace am was de e mined o be
1.56-100µg/mL and 0.39-100µg/mL o py idoxine hcl, acco dingly and we e shown co ela ion coe icien o 0.99. The p oposed
me hod exhibi ed signi ican p edic abili y and obus ness and showed pe cen age eco e y o 95.46% and 101.14%, espec i ely.
[16]
Padmaja, e .al in 2018 epo ed a RP-HPLC me hod o he simul aneous de e mina ion o me hyl cobalamin, alpha-lipoic acid,
py idoxine hyd ochlo ide, and olic acid d ugs. The column p e e ed was Ine sil C18 wi h dimensions 250mm × 4.6mm and pa icle
size o 5µm. The mobile phase was made up o ace oni ile: bu e mix u e (5.05 g o hexane-1-sul onic acid in 1000 mL o dis illed
wa e ) o e icien sepa a ion o sample a a a e o low o 1ml/min. The analysis was ca ied ou a con ex u e empe a u e and
de ec ed a 285nm. I was ound ha he a e age e en ion ime o me hyl cobalamin, py idoxine hyd ochlo ide, alpha-lipoic acid,
and olic acid we e 3.5, 6.7, 8.5, and 9.3 min wi h co ela ion coe icien o 0.99 o all he d ugs whe e he concen a ion anging om
0-2130 µg/mL (me hyl cobalamin), 0-142.5µg/mL (alpha-lipoic acid), 0-4.54µg/mL (py idoxine HCl) and 0-2µg/mL ( olic acid)
espec i ely. The eco e y a e o he de eloped me hod was ound o be 98-100%. [17]
N.M. Habib, e .al in 2017 in o de o de e mine a pai o mixed compounds con aining py idoxine HCl (PYH) wi h ei he meclizine
HCl (MEH) o cyclizine HCl (CYH), HPLC-DAD and TLC-densi ome ic echniques we e de eloped. Ch oma og aphic sepa a ion
o he h ee medica ions unde s udy was pe o med in he de ised TLC-densi ome ic me hod on silica gel 60 F254 pla es using a
mobile phase ha con ained me hylene chlo ide: ace one: me hanol (7: 1: 0.5, / / ) while he de ised HPLC-DAD me hod elied on
column (Zo bax Eclipse C18) wi h mobile phase (me hanol and 0.05M KH2PO4 in 90:10 / ; pH 5) a a low a e o 1mL/min whe e
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i was measu ed a 220nm. PYH, CYH and MEH we e ound o ha e R o 2.52, 4.4 and 7.65min acco dingly. The linea i y ange was
de e mined o be 10-50µg/mL (PYH), 10-50µg/mL (CYH) and 7-50µg/mL (MEH) espec i ely, wi h excellen co ela ion o 0.9999.
The me hod showed ema kable pe cen age eco e y (100% and 99%) and also used e ec i ely o iden i y he abo e-men ioned d ugs
in hei o mula ions. [18]
Ronald Ba za , e .al in 2016 u ilized HPLC me hod in an isoc a ic en i onmen and de ec ed a 290 nm by UV o examine i -B6
in comme cialized nu ien d inks and solid able s. C-18 column was used and he samples we e p epa ed using 81% dis illed wa e
and 19% e hanol. A e solubiliza ion in aqueous s a e wi h e hanol anging om 10-20% ( / ), i -B6 was iden i ied a 290 nm. A
cons an elu ion peak o B6 i amin was ob ained a 1.6 min a a e o low o 1.0 mL/minu e when he column p essu e was se a
1900 psi. The indings showed ha i amin B6 le els we e highly sensi i e a 290 nm, anging om 4.4029x10ˉ5 mola o 7.8081x10ˉ4
mola . Wi h a coe icien o de e mina ion o 0.9948 and an ex emely posi i e co ela ion coe icien o 0.9974, s anda d cu es
showed good linea i y in he ange o 0 o 7.8081x10ˉ4 mola (y =112,521,145.5x + 2,818.6) and he pe cen age eco e y o i amin
B6 a ied om 95% o 105%. [19]
Kamepalli Sujana, e .al in 2016 de eloped and alida ed a s aigh o wa d, eliable, ocused, and mo e p ecise UV spec oscopy
echnique o de e mining Py idoxine HCl, Folic acid, and Mecobalamin in bulk pha maceu icals and ma ke ed o mula ions
(capsules) employing he simul aneous equa ion app oach. The echnique en ails sol ing simul aneous equa ions by aking sol en
0.1N NaOH. The abso bance maxima (λmax) o py idoxine HCl, olic acid, and mecobalamin was ob ained a 218nm, 256nm, and
220nm, acco dingly. The echnique was alida ed in acco dance wi h ICH guidelines, and he calib a ion cu es o all h ee d ugs
showed high linea i y, as demons a ed by he co ela ion coe icien s ( ) o 0.999. Py idoxine HCl, olic acid, and mecobalamin we e
eco e ed in pe cen ages o 97.8%, 97.7%, and 91.76%, espec i ely. [20]
Adam, e .al in 2015 de eloped e e se phase HPLC which was quick, cos -e ec i e, eliable and speci ic o es ima ing py idoxine
hyd ochlo ide and i s b oken-down me aboli es. The analysis was ca ied ou using sol en sys em con aining 0.015M KH2PO4:
me hanol (70:30 / , pH- 3) and pe o med on The mo-hype sil GOLD C18 column wi h a low a e o 1mL/min o e icien sample
sepa a ion, de ec ion was ca ied ou a 254nm. A 3.5 minu es ± 0.02 he sepa a ion was inished, and no in e e ence om any
excipien s was seen. The me hod was es ima ed o be linea in he concen a ion anged om 10-50µg/ml wi h co ela ion o 0.9996
and pe cen age eco e y anged om 98.8 o 100.86%. The de eloped me hod was e ec i ely used o es ima ing he abo e-
men ioned d ugs and in hei combined o m. [21]
Kha eeb, e .al in 2015 de eloped a p ecise, e ec i e and speci ic spec opho ome ic me hod o an es ima ion o B-g oup i amins
such as i B3 (nico inamide) and i B6 (py idoxine HCl) in pha maceu ical p oduc s and in hei pu e o m. The echnique elies on
he i-iodide ions which we e o med when i amins eac wi h an amalgam o po assium iodide and ioda e a 25°C in an aqueous
solu ion and de ec ed o i B6 a 290 and 335nm and a 288 and 350nm o i B3 espec i ely. The p oposed me hod was alida ed
in e ms o linea i y, accu acy, p ecision, LOD and LOQ. The esul s we e ound o be linea in he concen a ion ange o 0.5-20µg/mL
o i B3 and i B6 wi h s ong co ela ion coe icien ( 2= 0.998:0.9949, 0.9995:0.9974) and wi h pe cen age eco e y anges
be ween 96% o 102.25% espec i ely, indica ing ha he excipien s in o mula ion did no in e e e wi h he i amins. [22]
Khanage SG and cowo ke s in 2014 used a RP-HPLC me hod o he es ima ion o P ochlo pe azine Malea e (PCM) and Py idoxine
hyd ochlo ide (PDH) in pha maceu ical p oduc s. Mobile phase used was 40:60 / o me hanol: wa e a pH 7 wi h a a e o low
o 1 mL/min, HPLC was pe o med on a C18 column and e luen was measu ed a 272 nm. I was ound ha he e en ion imes o
PDH and PCM we e 3.48 and 6.28 minu es espec i ely and he linea i y was de e mined in he ange o 5-25 μg/ml. This app oach
e ec i ely analysed able dosage o ms, wi h no ch oma og aphic in e e ences om o mula ion excipien s. The analysis o PCM
and PDH in able s quan i a i ely was e ec i ely conduc ed in his s udy using he HPLC echnique, which is easy o use, quick, ee
om in e e ence om o mula ion excipien s and doesn' equi e a sepa a ion s ep o each medica ion. [23]
T. Jeyalakshmi, e .al in 2014 iden i ied accu a e, easy o use, and sensi i e e e se phase-HPLC me hod o simul aneously
es ima ing i amin B1 (Thiamine hyd ochlo ide), i amin B3 (Nico inamide), i amin B5 (Dexpan henol) and i amin B6(py idoxine
hyd ochlo ide). The sample sepa a ion was ca ied ou on Wa e C18 column (250mm × 4.6mm, 5µm). The mobile phase composed
o bu e and me hanol (95:5 / , pH 3.5) was used o e icien sepa a ion a a low a e o 1.5mL/min and he mobile we e allowed
o se le in he column o 15min and measu ed a 210nm. Thiamine HCl, Nico inamide, Dexpan henol and Py idoxine HCl we e
ound o ha e e en ion ime o 2.492min, 6.748min, 20.08min, 4.077min and showed linea i y in he ange o 10-200µg/ml, 40-
800µg/ml, 8-160µg/ml wi h coe icien co ela ion o 0.9992, 0.9994, 0.9993 and 0.9991 acco dingly. The p ecision pe cen age
ela i e o he s anda d de ia ion eme ged was below 2% and ound he app oach was speci ic ha could be used o ou ine analyses
o he d ug p oduc s. [24]
Shinde P ashan i, e .al in 2014 A simple, selec i e, apid, p ecise and economical RP-HPLC me hod has been de eloped o he
de e mina ion o DL-Me hionine and Py idoxine Hyd ochlo ide in able o mula ion. Po assium dihyd ogen o hophospha e was used
as he mobile phase a a low a e o 1 mL/min on an isoc a ic ha included an Agilen 1200 HPLC sys em wi h a a iable wa eleng h
UV de ec o wi h a Pee less basic C18 (4.6 mm x 15 cm, 5 μm) column a 210 nm. Py idoxine HCl and DL-me hionine we e ound
o ha e he e en ion ime 14 min and 3.5 min and pe cen age eco e y ound anging om 98% o 102%. The es ablished app oach
was app op ia e o egula d ug analysis in able o m o dosage since i was mo e accu a e, s aigh o wa d and ep oducible. [25]
G. Nagamallika, e .al in 2013 Fo he pu pose o quan i a i ely de e mining he le els o hiamine hcl, nico inamide, ibo la in-5′-
phospha e sodium, py idoxine hyd ochlo ide, ca eine, D (+)-Pan henol, and p ese a i es such as me hylpa aben and p opylpa aben
in mul i i amin sy up o mula ion, a new, sensi i e, and highly selec i e g adien e e se phase UPLC echnique was iden i ied and
alida ed. The sepa a ion was ob ained by u ilizing sol en A (0.1% TFA in wa e ) and sol en B (a combina ion o 50% ace oni ile
and 50% me hanol wi h 1.0 mL/ min ), on an HSS T3 (50mm × 2.1mm, 1.7μm) column by main aining empe a u e o he column a
48°C and de ec ed a 200, 254 and 290 nm. Fo hiamine hcl, py idoxine hyd ochlo ide, ca eine, Nico inamide, ibo la in-5′-
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phospha e sodium, D (+) Pan henol, me hylpa aben, and p opylpa aben, he co ela ion coe icien was 0.99. The p oduc was
subjec ed o s ess ul condi ions including oxida i e, acidic, basic, hyd oly ic, he mal, and pho oly ic deg ada ion in o de o
demons a e he me hod's s abili y-indica ing capabili ies. Thiamine hyd ochlo ide, py idoxine hyd ochlo ide, D (+)-Pan henol,
Nico inamide, ibo la in-5′-phospha e sodium, ca eine, me hylpa aben, and p opylpa aben we e among he b eakdown p oduc s ha
could be clea ly sepa a ed. The me hod's speci ici y, linea i y, accu acy, p ecision, and obus ness we e all alida ed in acco dance
wi h ICH guidelines. [26]
Reema, e .al in 2013 de eloped a p ecise, accu a e and e ec i e RP-HPLC me hod o es ima ing and alida ing i amin B d ugs
such as py idoxine hyd ochlo ide (B6), olic acid (B9), me hyl cobalamin (B12) and a o as a in. Due o he low concen a ion o
me hyl cobalamin (B12) in able s, i s de e mina ion was done independen ly. The sepa a ion was achie ed using mobile phase 100%
me hanol o i -B12 while phospha e bu e and 100% ace oni ile o es ima ing i -B6, B9 and a o as a in (50:50 / , pH-3). Ine sil
ODS C18 column wi h dimension (4.6 x 250mm) and 5µm pa icle size packing was used o he sample sepa a ion wi h a un ime
o 25min and de ec ed a 265nm and 254nm. The e en ion ime was de e mined o be 2.358min o i B9, 2.516min o i B12,
1.952min o i B6 and 10.837min o a o as a in. In he concen a ion ange o 25–250 ppm (ATO), 50–500 ppm ( i amin B6), 10-
100 ppm ( i amin B9), and 2–50 ppm ( i amin B12), he esul s appea ed o be linea wi h co ela ion o 0.999 espec i ely. The a e
o eco e y was de e mined o be 101.14, 101.02, 101.22, 101.66 acco dingly, which made he me hod mo e accu a e and speci ic.
[27]
Nayak, e .al in 2013 in o de o es ima e PYR (py idoxine HCl) and DOX (doxylamine succina e) simul aneously in a able dose
o m, an easy- o-use, quick UV spec opho ome ic echnique was de eloped. Resea ch on solubili y was done o ind a good sol en
ha would dissol e bo h PYR and DOX. Subsequen ly, he medica ions we e dissol ed sepa a ely and in combina ion in he sol en
and he abso bances o PYR and DOX we e de e mined a 260 nm and 290 nm, co espondingly and ollowed Bee -Lambe ’s law
wi hin he concen a ion ha anged om 4-20µg/ml. Valida ion was done in acco dance wi h ICH guidelines using a iuos
pa ame e s. The p oposed echnique showed excellen accu acy wi h eco e y a es anging om 99.41% o 100.18%, a s ong
co ela ion coe icien o 0.999 and possessed low LOD and LOQ alues indica ing he echnique was highly sensi i e owa ds
de e mining he ci ed d ugs in a la ge quan i y and in able dose o m. [28]
Ma cin Leszek Ma szall, e .al in 2005 epo ed a echnique employing high pe o mance liquid ch oma og aphy (HPLC) wi h
coulome ic elec ochemical and UV de ec o o he simul aneously measu ing hiamine hyd ochlo ide, py idoxine hyd ochlo ide,
and cyanocobalamin. Vi amin e en ion imes we e egula ly measu ed by isoc a ic elu ion using a Supelco LC 18 column measu ing
5 μm (25 cm×4.6 mm) and a mobile phase o 0.05M phospha e bu e , 10% me hanol and 0.018M ime hylamine (1mL/min, pH
3.55). Re en ion cha ac e is ics, coulome ic elec ochemical analysis, and ul a iole de ec ion all p o ed he me hod's speci ici y.
The de ec ion limi s o he ci ed d ugs (py idoxine, hiamine, and cyanocobalamin de ec ion limi s we e 2.7, 9.2, and 0.08 ng/ml,
co espondingly. La ge concen a ion ange, high sensi i i y, and adequa e accu acy (99.6–102.7%) we e u he cha ac e is ics o his
app oach. When he me hod's epea abili y was assessed a a ious i amin concen a ions, he ela i e s anda d de ia ion was less
han 4.5%. The echnique has been e ec i ely used o quan i y he le els o i amins like B1, B6, and B12 in die a y supplemen s and
medicinal o mula ions. [29]
CONCLUSION
In his comp ehensi e su ey, we ha e e iewed a wide a ay o analy ical me hods employed o he measu emen and e alua ion o
py idoxine hyd ochlo ide ( i amin B6) and i s combina ion he apies. Py idoxine hyd ochlo ide plays a c ucial ole in a ious
enzyma ic pa hways in ol ed in amino acid me abolism, neu o ansmi e syn hesis, and ene gy p oduc ion. As i s signi icance
expands om solo o mula ions o complex combina ion he apies, i becomes impe a i e o employ ad anced analy ical echniques
o ensu e he quali y, sa e y, and e icacy o pha maceu ical o mula ions. A numbe o s udies ha e in es iga ed he use o a ious
analy ical echniques, including RP-HPLC, LC-MS/MS, UV spec oscopy, and high-pe o mance liquid ch oma og aphy (HPLC), o
quan i y py idoxine hyd ochlo ide alone o in combina ion wi h o he medica ions. These echniques p o ide excellen sensi i i y,
speci ici y, accu acy, and p ecision o igu ing ou how much py idoxine hyd ochlo ide is p esen in a ious d ug o mula ions.
Fu he mo e, hese echniques accep abili y and dependabili y o egula analysis in pha maceu ical labs a e gua an eed by hei
alida ion in acco dance wi h In e na ional Con e ence on Ha moniza ion (ICH) equi emen s. Se e al dosage o ms, including
able s, nu i ional be e ages, and mul i i amin sy ups, ha e been success ully analysed using hese echniques, p o ing hei
adap abili y and sui abili y o use in a a ie y o o mula ions.
In conclusion, hese me hodologies no only con ibu e o he de elopmen o high-quali y pha maceu ical p oduc s bu also acili a e
he e ec i e managemen o a ious heal h condi ions whe e py idoxine hyd ochlo ide supplemen a ion is wa an ed. Fu u e esea ch
in his ield should ocus on u he op imiza ion and alida ion o analy ical me hods o mee he e ol ing needs o he pha maceu ical
indus y and heal hca e p ac i ione s.
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