Chemometric-Assisted Hydrophilic Interaction Chromatographic Method for the Determination of Gadolinium-Based Magnetic Resonance Imaging Contrast Agent in Liposomes

Maia, Ana Luiza C.; Silva, Pedro Henrique R. da; Fernandes, Christian; Silva, Aline T. M. e; Barros, André Luís B. de; Soares, Daniel Crístian F.; Ramaldes, Gilson A.

doi:10.21577/0103-5053.20180120

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Article • J. Braz. Chem. Soc. 29 (11) • Nov 2018 • https://doi.org/10.21577/0103-5053.20180120 copy

Chemometric-Assisted Hydrophilic Interaction Chromatographic Method for the Determination of Gadolinium-Based Magnetic Resonance Imaging Contrast Agent in Liposomes

Authorship SCIMAGO INSTITUTIONS RANKINGS

Gadodiamide (Gd-DTPA-BMA) is a gadolinium (Gd) chelate composed of two carboxylate groups of diethylenetriaminepentaacetic acid (DTPA) and two amide groups (BMA). Gd complexes are the most widely used contrast agents in nuclear magnetic resonance. Furthermore, our research group has demonstrated the potential of liposomes containing Gd-DTPA-BMA for cancer therapy. The aim of this study was to develop and validate a chemometric-assisted method by hydrophilic interaction liquid chromatography (HILIC) for determination of Gd-DTPA-BMA in liposomes. The chromatographic conditions obtained were: Sequant® ZIC®-HILIC Merck (150 × 4.6 mm, 3.5 µm, 100 Å) column, mobile phase composed of 5 mmol L^-1 ACN/NH₄FA, pH 4.5 (60:40 v/v) at 0.6 mL min^-1, injection volume of 20 µL, temperature of 30 ºC, and detection at 210 nm. The linear range was of 40 to 120 nmol mL^-1. The use of chemometrics allowed obtaining optimal chromatographic parameters, in terms of signal-to-noise ratio, resolution, and asymmetry.

Keywords:
gadodiamide; liposomes; hydrophilic interaction chromatography; chemometrics; Box-Behnken

Experiment	Parameter evaluated	Independent variable	Mobile phase composition
1	type of buffer	NH₄Ac	ACN/NH₄Ac 10 mmol L^-1, pH 5.8, 70:30 v/v
2	type of buffer	NH₄FA	ACN/NH₄FA 10 mmol L^-1, pH 4.7, 70:30 v/v
3	ACN ratio	60%	ACN/NH₄FA 10 mmol L^-1, pH 4.7, 60:40 v/v
4		70%	ACN/NH₄FA 10 mmol L^-1, pH 4.7, 70:30 v/v
5		75%	ACN/NH₄FA 10 mmol L^-1, pH 4.7, 75:25 v/v
6	buffer concentration	5 mmol L^-1	ACN/NH₄FA 5 mmol L^-1, pH 4.7, 70:30 v/v
7		10 mmol L^-1	ACN/NH₄FA 10 mmol L^-1, pH 4.7, 70:30 v/v
8		15 mmol L^-1	ACN/NH₄FA 15 mmol L^-1, pH 4.7, 70:30 v/v
9	the aqueous phase pH of the mobile phase	2.7	ACN/NH₄FA 10 mmol L^-1, pH 2.7, 70:30 v/v
10		3.7	ACN/NH₄FA 10 mmol L^-1, pH 3.7, 70:30 v/v
11		4.7	ACN/NH₄FA 10 mmol L^-1, pH 4.7, 70:30 v/v

Analytical parameter	Condition		Factorial combination
Analytical parameter	Nominal	Varied	1	2	3	4	5	6	7	8
ACN ratio in the mobile phase / %	60 (A)	63 (a)	A	A	A	A	a	a	a	a
The aqueous phase pH of the mobile phase	4.5 (B)	4.7 (b)	B	B	b	b	B	B	b	b
Buffer concentration / (mmol L^-1)	5 (C)	5.5 (c)	C	c	C	c	C	c	C	c
Column temperature / ºC	30 (D)	33 (d)	D	D	d	d	d	d	D	D
Flow-rate / (mL min^-1)	0.6 (E)	0.7 (e)	E	e	E	e	e	E	e	E
ACN brand	Tedia (F)	J.T. Baker (f)	F	f	f	F	F	f	f	F
Buffer brand	Vetec (G)	Spectrum (g)	G	g	g	G	g	G	G	g

Independent variable					Level
Independent variable					-1		0	1
X₁	pH				3.7		4.2	4.7
X₂	ACN ratio / %				60		65	70
X₃	buffer concentration / (mmol L^-1)				5		15	25
Experiment	Contrast			Experimental condition			Dependent variable^a a Values are expressed as mean of 3 injections;
Experiment	X₁	X₂	X₃	X₁	X₂	X₃	Signal-to-noise ratio	R_s	A_s
1	1	1	0	4.7	70	15	7164899	7.7	1.32
2	1	-1	0	4.7	60	15	7846412	3.7	1.18
3	-1	1	0	3.7	70	15	4029493	1.3	1.31
4	-1	-1	0	3.7	60	15	6040335	0.0	1.21
5	1	0	1	4.7	65	25	1675031	5.1	1.31
6	1	0	-1	4.7	65	5	9622069	5.5	1.25
7	-1	0	1	3.7	65	25	6731611	0.0	1.27
8	-1	0	-1	3.7	65	5	7473207	4.0	1.25
9	0	1	1	4.2	70	25	2324785	7.2	1.30
10	0	1	-1	4.2	70	5	5124660	7.2	1.33
11	0	-1	1	4.2	60	25	3487648	2.7	1.23
12	0	-1	-1	4.2	60	5	6514122	3.5	1.19
13^b b values are expressed as mean (n = 3 samples, being 3 injections for each sample); ,^c c central point. Rs: resolution; As: asymmetry.	0	0	0	4.2	65	15	2293381	3.7	1.27

Linear range / %	Gd-DTPA-BMA mean concentration ± SD / (nmol mL^-1)			RSD / %			Accuracy result
	Intra-day^a a Mean of three determinations;		Inter-day^b b mean of six determinations; cmean of nine determinations. Gd-DTPA-BMA: gadodiamide; SD: standard deviation; RSD: relative standard deviation.	Intra-day^a a Mean of three determinations;		Inter-day^b b mean of six determinations; cmean of nine determinations. Gd-DTPA-BMA: gadodiamide; SD: standard deviation; RSD: relative standard deviation.	Gd-DTPA-BMA amount added / (nmol mL^-1)	Gd-DTPA-BMA mean concentration^a a Mean of three determinations; ± SD / (nmol mL^-1)	Mean recovery^a a Mean of three determinations; ± SD / %
	Day 1	Day 2		Day 1	Day 2		Gd-DTPA-BMA amount added / (nmol mL^-1)		Mean recovery^a a Mean of three determinations; ± SD / %
50	39.59 ± 0.41	38.82 ± 0.52	39.20 ± 0.59	1.05	1.33	1.51	40	38.93 ± 0.37	97.32 ± 0.93
100	79.80 ± 0.86	79.13 ± 0.23	79.47 ± 0.67	1.08	0.29	0.85	80	79.41 ± 0.31	99.26 ± 0.38
150	119.50 ± 0.60	119.49 ± 0.94	119.50 ± 0.71	0.50	0.79	0.59	120	119.10 ± 1.97	99.25 ± 1.65
Mean^c									98.61 ± 1.37
RSD^c / %									1.38

Variable	Content^a a Mean values obtained at nominal conditions subtracted from the mean values obtained under the varied conditions; / %	R_s^a a Mean values obtained at nominal conditions subtracted from the mean values obtained under the varied conditions;	A_s^a a Mean values obtained at nominal conditions subtracted from the mean values obtained under the varied conditions;
ACN ratio in the mobile phase (A = 60%; a = 63%)	101.37 - 100.87 = 0.50	3.21 - 4.57 = -1.36	1.32 - 1.28 = 0.04
The aqueous phase pH of the mobile phase (B = 4.5; b = 4.7)	100.61 - 101.64 = -1.03	4.06 - 3.72 = 0.34	1.26 - 1.34 = -0.08
Buffer concentration (C = 5 mmol L^-1; c = 5.5 mmol L^-1)	101.46 - 100.78 = 0.68	3.56 - 4.22 = -0.66	1.29 - 1.30 = -0.01
Column temperature (D = 30 ºC; d = 33 ºC)	101.35 - 100.90 = 0.45	3.80 - 3.98 = -0.18	1.29 - 1.31 = -0.02
Flow (E = 0.6 mL min^-1; e = 0.7 mL min^-1)	101.16 - 101.09 = 0.07	4.37 - 3.41 = 0.96	1.28 - 1.31 = -0.03
ACN brand (F = Tedia; f = J.T.Baker)	101.54 - 100.70 = 0.84	3.83 - 3.95 = -0.12	1.25 - 1.35 = -0.10
Buffer brand (G = Vetec; g = Spectrum)	101.26 - 100.98 = 0.28	4.05 - 3.73 = 0.32	1.26 - 1.34 = -0.08
Largest effect^b b RSD (relative standard deviation) between the values obtained in the 8 experiments multiplied by root of 2. Rs: resolution; As: asymmetry.	1.24	36.06	8.97

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