Mixed-Mode Liquid Chromatography (MMLC) includes several separation
mechanisms in a single column, which is why MMLC can analyze compounds in a broad
range of polarities and ionization potentials in a single run (1). Acclaim Mixed-Mode WCX-1
column with the ability to expose hydrophobic and weak cation exchange interactions was
thus selected to analyse a challenging mixture of neutral and cationic forms: ergotamine,
mecloxamine, camylofin, caffeine and propyphenazone, used as a fixed combination. MMLC
method was developed in line with Analytical Quality by Design (AQbD) approach implying
the scientifically-based understanding of process properties and risk-based management of
the method life cycle. AQbD refers to pre-definition of the method’s Analytical Target Profile
(ATP) by means of baseline separations within the shortest possible time, as well as
definition of Critical Method Attributes (CMAs) as a measure of method quality and Critical
Method Parameters (CMPs) affecting CMAs (2). Acetonitrile content, pH and acetate buffer
concentration were selected as CMPs since retention mechanism expression in MMLC
strongly depends on the mobile phase characteristics. The dependence of CMAs on CMPs was
revealed following a face-centred central composition design plan of experiments and
accompanying mathematical models, coefficients and standard error values. Design Space in
which ATP is achieved with a high level of reliability (π = 90%), was determined by Monte
Carlo simulations taking error distribution into account. Its margins pointed out to the
working point that assures proper method robustness (pH 5.2, 90 mM acetate buffer solution
and 48% (v/v) of acetonitrile).Multimodalna tečna hromatografija (Mixed‐Mode Liquid Chromatography – MMLC)
uključuje nekoliko mehanizama razdvajanja u jednoj koloni, zbog čega se ova tehnika može
koristiti za simultanu analizu jedinjenja širokog opsega polarnosti i jonizacionog potencijala
(1). Acclaim Mixed‐Mode WCX‐1 kolona sa sposobnošću ekspresije hidrofobnih i interakcija
slabe katjonske izmene, je odabrana za analizu izazovne smeše neutralnih i katjonskih oblika
analita: ergotamina, mekloksamina, kamilofina, kofeina i propifenazona, koji se primenjuju u
fiksnoj kombinaciji. MMLC metoda je razvijena u skladu sa pristupom ugradnje kvaliteta kroz
dizajn (Analytical Quality by Design – AQbD) koji podrazumeva naučno zasnovano
razumevanje svojstava procesa i upravljanje životnim ciklusom metode prema riziku. AQbD
se odnosi na unapred definisanje analitičkog ciljanog profila metode (Analytical Target
Profile - ATP) odnosno razdvajanje na baznoj liniji za što kraće vreme, kao i na definisanje
kritičnih osobina metode (Critical Method Attributes - CMA) kao mere kvaliteta metode i
kritičnih parametara metode (Critical Method Parameters - CMP) koji utiču na CMA (2).
Sadržaj acetonitrila, pH i koncentracija acetatnog pufera izabrani su kao CMP, pošto
ekspresija MMLC retencionih mehanizma zavisi od karakteristika mobilne faze. Zavisnost
CMA od CMP definisana je pomoću plana eksperimenata usklađenim sa centralnim
kompozicionim dizajnom, ka centru orijentisanim i pratećim matematičkim modelima,
koeficijentima i vrednostima standardne greške. Prostor dizajna u kome se ATP postiže sa
visokim nivoom pouzdanosti (π = 90%) određen je Monte Karlo simulacijama uzimajuć i u
obzir distribuciju grešaka. Njegov okvir ukazuje na radnu tačku koja obezbeđuje
odgovarajuć u robusnost metode (pH 5,2, 90 mM rastvor acetatnog pufera i 48% (v/v)
acetonitrila).VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra
