Analysis of a second-generation tetracycline antibiotic minocycline in human plasma by LC–MS/MS

Methodology

Tetracyclines (TCs) are an important group of antibiotics, used commonly in the treat-ment of infectious diseases and as an additive to animal feeds for their broad spectrum anti-bacterial activity and cost–effectiveness [1,2]. Minocycline (MC) is a semi-synthetic second-generation TC antibiotic and is primarily used in the treatment of acne vulgaris [3,4]. It is the most lipid soluble of the TC class of antibiotics, gets absorbed rapidly and readily crosses the blood–brain barrier [5]. Although it has broad-spectrum antibacterial activity, it shows bio-logical effects that are fundamentally different from its antimicrobial action. Several studies have reported the potential use of MC as a cyto-protectant in the treatment of several neuro-logical disorders, including amyotrophic lateral sclerosis [6], multiple sclerosis [7], Alzheimer’s disease [8], Huntington’s disease [9], Leber’s hereditary optic neuropathy [10] and Parkinson’s disease [11]. The bioavailability of MC is almost 100% and is mainly excreted through faeces. Similarly to other TCs, MC is an unstable com-pound and is prone to degrade under acidic (pH 2.0–6.0) and light conditions. The degra-dation products are formed via epimerization, dehydration and proton transfer pathways. For TCs without a hydroxyl group at C-5 position, 4-epimerization is favored in acidic to neutral conditions. This process leads to the forma-tion of 4-epiminocycline (4-EMC), a potential degradation product and a major impurity of MC [12].

Several methods based on spectrofluorimetry [13], microbiological assay [13], semi-differential cyclic voltammetry [14], luminescence [15], HPTLC [16], HPLC [17–33], LC–TOF–MS [33], UPLC–TOF-MS [34] and LC–MS/MS [35,36], have been reported to determine MC in a vari-ety of matrices. These include honey samples [33], environmental water samples [36], discolored teeth [25], saliva [16,28], gingival fluid [16], muscle and liver of porcine, chicken and bovine [32], pig tissue [26], brain tissue [29,31], pig serum [26], mice serum [24], human serum [13,15], urine [15,34], rat plasma [29,31] and human plasma [16,27,28,35]. Wrightson and co-workers [26] have analyzed MC in pig tissue and serum with a limit of quan-titation of 100 ng/ml and a chromatographic runtime of 15 min. A rapid method to deter-mine MC in pharmaceutical formulations and human urine/serum has been proposed based on the MC-europium-sodium dodecylbenzene sulfonate luminescence system [15]. A linear rela-tionship was established over the range of 182–4575 ng/ml, with a LOD of 9 ng/ml. Tao et al. have demonstrated a new robust HPLC–UV method for the simultaneous determination of TCs in muscle and liver of porcine, chicken and bovine by accelerated solvent extraction method [32]. The detection limits for seven TCs varied from 4.6 to 306.4 µg/kg in different matrices. Nevertheless, five pertinent methods developed and validated for MC in plasma can be com-pared with the present work [27–29,31,35]. Mascher developed a sensitive and specific method to

Analysis of a second-generation tetracycline antibiotic minocycline in human plasma by LC–MS/MS

A simple, selective and sensitive LC–MS/MS assay has been developed for the determination of minocycline in human plasma. Plasma samples were prepared by protein precipitation, followed by chromatographic separation on a HyPURITY™ C8 (100 mm × 4.6 mm, 5 µm) column under isocratic conditions. The LOD and LLOQ of the method were 0.50 and 20.0 ng/ml, respectively. The intra-batch and inter-batch precision (% coefficient of variation) across quality control levels was less than 8.0%. For a set of incurred samples the percentage change in concentration was within ± 9.0%. The method was successfully applied to a bioequivalence study of 135 mg minocycline tablet formulation in 14 healthy Indian males. The reproducibility in the measurement of study data was demonstrated by incurred sample reanalysis.

Dinesh S Patel1,2, Naveen Sharma2, Mukesh C Patel1, Bhavin N Patel2,3, Pranav S Shrivastav†3 & Mallika Sanyal4

1Chemistry Department, Pramukh Swami Science and H.D. Patel Arts College, Sarva Vidyalaya Campus, Kadi-382715, Gujarat, India 2Bio-Analytical Laboratory, BA Research India Ltd., Bodakdev, Ahmedabad-380 054, Gujarat, India 3Department of Chemistry, School of Sciences, Gujarat University, Navrangpura, Ahmedabad-380009, Gujarat, India 4Department of Chemistry, St. Xavier’s College, Navrangpura, Ahmedabad-380009, Gujarat, India †Author for correspondence:Tel.: +91 079 2630 0969 Fax: +91 079 2630 8545 E-mail: pranav_shrivastav@ yahoo.com

2177ISSN 1757-6180Bioanalysis (2011) 3(19), 2177–219410.4155/BIO.11.226 © 2011 Future Science Ltd

For reprint orders, please contact [email protected]

determine MC in human plasma by HPLC–UV [27]. The plasma samples were prepared by liquid–liquid extraction (LLE) in ethyl acetate and the linearity of the assay was established in the range of 28–3533 ng/ml. However, the method was not applied to real sample ana lysis. Similarly, three other less sensitive HPLC–UV-based methods [29,28,31] have been described with LOQ ranging from 50–150 ng/ml in rat and human plasma, having a chromatographic run time of ≥15 min. A sensitive (5 ng/ml) and rapid (3.0 min) method has been described to determine MC in human plasma by LC–MS/MS [35]. This method was examined to check the drug–food interaction and applied to a bioequivalence study in 24 healthy volunteers. Although the method is promising for routine subject sample ana lysis, however, studies related to matrix effect, ion suppression, stability issue and incurred sample reana lysis of MC were not conversed. A detailed comparison of salient fea-tures of methods developed for MC in plasma is shown in Table 1.

Thus, in the present study a simple, sensitive and selective LC–MS/MS method has been pro-posed for the determination of MC in human plasma. The analyte and internal standard (IS) were extracted via protein precipitation with acetonitrile and separated on a HyPURITY™ C8 column within 4.5 min. Under the opti-mized isocratic conditions, it was possible to separate, chromatographically, 4-EMC and 4-epitetracycline (4-ETC), the major impuri-ties in MC and IS, respectively. The method was extensively validated as per the US FDA guide-lines. The assay developed is highly rugged and adequately sensitive for routine subject sample ana lysis. The method was successfully applied to a bioequivalence study of 135 mg MC tablet formulation in 14 healthy Indian male subjects under fasting conditions.

Experimental � Chemicals & materials

Minocycline hydrochloride (Catalog no. 1444004) was a US Pharmacopeia (USP) ref-erence standard, while TC hydrochloride (IS, 95.8%) was purchased from Vivan Life Sciences Pvt. Ltd (Mumbai, India). HPLC grade metha-nol and acetonitrile were procured from SD Fine Chemicals Ltd (Mumbai, India). Analytical grade formic acid (90.0%) was procured from SD Fine Chemicals Ltd and ammonium salt of trifluoroacetic acid (ATFA, 98%) was obtained from Acros Organics (NJ, USA). Water used for

LC–MS/MS was prepared using Milli Q water purification system from Millipore (Bangalore, India). Control human plasma with K3-EDTA as anticoagulant was procured from BA Research India Ltd (Ahmedabad, India) and was stored at -70°C. Peerless Basic C18 (100 × 4.6 mm, 3 µm) was purchased from Chromatopak Analytical Instrumentation Pvt. Ltd. (Mumbai, India), while HyPURITY Cyano (50 × 4.6mm, 5 µm), BetaBasic CN (100 × 2.1 mm, 5 µm) and HyPURITY C8 (100 × 4.6 mm, 5 µm) were procured from Thermo Scientific (NJ, USA). Centrifuge was of Eppendorf 5810 (Hamburg, Germany). Gold covRguard lamp, F40T12 Gold CVG SPD for blocking UV light was purchased from General Electric (MO, USA). The dilution solution was methanol:deionized water at a ratio of 70:30, v/v.

� LC–MS/MS instrumentation & conditionsThe liquid chromatography system from Shimadzu (Kyoto, Japan) consisted of a LC-10ADvp pump, an autosampler (SIL-HTc) and an online degasser (DGU-14A). The chro-matographic column used was HyPURITY C8 (100 mm length × 4.6 mm inner diameter, with 5.0 µm particle size) from Thermo Scientific and maintained at 30°C in a column oven. The mobile phase consisted of 0.05% formic acid and 0.01%, 1M ATFA solution in deionized water:methanol (70:30, v/v). Separation of MC and IS was performed under isocratic conditions at a flow rate of 0.8 ml/min. The autosampler temperature was maintained at 4°C and the injection volume was kept at 5.0 µl. The total LC run time was 4.5 min. The total eluant from the column was split in a 20:80 ratio; flow directed to the ion spray interface was equivalent to 160 μl/min.

Ionization and detection of analyte and IS was performed on a triple quadrupole mass spectrometer, API-4000 equipped with Turbo Ion spray®, from AB SCIEX (Toronto, Canada) operating in the positive ion mode. Quantitation was done using MRM mode to monitor pro-tonated precursor → product ion transition at m/z 458.2 → 441.2 for MC and 445.2 → 410.1 for IS (Figure 1a & b). All the parameters of LC and MS were controlled by Analyst software version 1.4.2.

For MC and IS the source dependent param-eters maintained were gas 1 (nebulizer gas): 50 psi, gas 2 (heater gas): 55 psi, ion spray volt-age: 3500 V, turbo heater temperature: 550°C, entrance potential: 10 V, collision activation

Methodology | Patel, Sharma, Patel, Patel, Shrivastav & Sanyal

Bioanalysis (2011) 3(19)2178 future science group

Tab

le 1

. Su

mm

ary

of

anal

ytic

al m

eth

od

s fo

r d

eter

min

atio

n o

f m

ino

cycl

ine

in p

lasm

a.

Sr. N

o.

Extr

acti

on

pro

ced

ure

(b

iolo

gic

al

sam

ple

vo

lum

e); m

ean

rec

ove

ry;

inte

rnal

sta

nd

ard

Co

lum

n; e

luti

on

typ

e; m

ob

ile p

has

e;

flo

w r

ate

; in

ject

ion

vo

lum

eD

etec

tio

n; m

axim

um

on

-co

lum

n lo

adin

g a

t U

LOQ

per

in

ject

ion

vo

lum

e; a

nal

ytic

al r

un

tim

e; r

eten

tio

n t

ime

; lin

ear

dyn

amic

ran

ge

; ap

plic

atio

n

Ref

.

1Li

quid

–liq

uid

extr

actio

n w

ith 5

ml e

thyl

ac

etat

e, b

ack

extr

actio

n in

0.5

ml,

0.02

M

HC

l (1.

0 m

l, hu

man

pla

sma)

; 76.

4%; –

Nuc

leos

il 5

-CN

(125

mm

× 4

.0 m

m, 5

µm

);

isoc

ratic

; met

hano

l:20

mM

per

chlo

ric a

cid

/4 m

M t

rieth

ylam

ine

in w

ater

(20

:80,

v/v

);

1.0

ml/

min

; 20

µl

HPL

C–U

V (3

50 n

m);

30.

3 ng

; 4.0

min

; 2.8

min

; 28

–353

3 ng

/ml;

phar

mac

okin

etic

stu

dy in

a h

ealth

y vo

lunt

eer

[27]

2So

lid-p

hase

ext

ract

ion

on W

ater

s O

asis

HLB

ca

rtrid

ge (1

.0 m

l, hu

man

pla

sma

and

paro

tid s

aliv

a); 9

9.3%

in p

lasm

a an

d 91

.3%

in p

arot

id s

aliv

a; d

emec

locy

clin

e

Nuc

leos

il C

8 (2

50 m

m×

4.0

mm

, 5 µ

m);

is

ocra

tic; a

ceto

nitr

ile:m

etha

nol:w

ater

:0.1

%

trifl

uoro

acet

ic a

cid

(25:

2:72

.9:0

.1, v

/v/v

/v);

0.

9 m

l/m

in; 2

0 µl

HPL

C–U

V (3

50 n

m);

50

00

ng f

or p

lasm

a an

d pa

rotid

sal

iva;

15.

0 m

in; 4

.2 m

in; 1

00

–25,

00

0 ng

/ml

[28]

3Li

quid

–liq

uid

extr

actio

n w

ith 4

ml d

ieth

yl

ethe

r-di

chlo

rom

etha

ne (

0.2

ml,

hum

an

plas

ma)

; 86.

2%; c

larit

hrom

ycin

Zorb

ax R

X-C

8 (1

50 m

m ×

4.6

mm

, 5 µ

m);

is

ocra

tic; a

ceto

nitr

ile:w

ater

:trifl

uoro

ace

tic a

cid

(80

:19.

9:0

.1, v

/v/v

); 1

.0m

l/m

in; 4

0 µl

LC–M

S/M

S; 8

0 ng

; 3 m

in; 1

.77

min

; 5.0

–20

00

ng/m

l; bi

oequ

ival

ence

stu

dy in

24

heal

thy

volu

ntee

rs[35]

4So

lid-p

hase

ext

ract

ion

on W

ater

s O

asis

HLB

ca

rtrid

ge (

0.2

ml r

at p

lasm

a an

d 20

0 m

g br

ain

tissu

e); 8

9% in

pla

sma

and

81%

in

tissu

e; d

emec

locy

clin

e/te

trac

yclin

e

Sym

met

ry S

hiel

d RP

8 (1

50 m

m×

4.6

mm

, 3.

5 µm

); is

ocra

tic; a

ceto

nitr

ile:m

etha

nol:w

ater

: 0.0

1 M

KH

2PO

4 (5

:20

:72.

1, v

/v/v

) con

tain

ing

0.03

mM

Na 2E

DTA

and

60%

HC

lO4,

2.9

ml,

pH

2.5

with

10

mM

KO

H; 0

.8 m

l/m

in; 2

0 µl

HPL

C–U

V (3

50 n

m);

6.2

5 ng

; 30.

0 m

in; 1

0.1

min

; 50

–250

0 ng

/ml f

or p

lasm

a an

d br

ain

tissu

e; b

rain

–blo

od

plas

ma

dist

ribut

ion

stud

ies

[29]

5So

lid-p

hase

ext

ract

ion

on W

ater

s O

asis

HLB

ca

rtrid

ge (

0.15

–0.4

ml m

ice

plas

ma

dilu

ted

up t

o 1.

0 m

l with

hum

an p

lasm

a, 2

00

mg

brai

n tis

sue)

; 80%

in p

lasm

a an

d 4

0% in

br

ain

tissu

e; –

Sym

met

ry S

hiel

d RP

8 (2

50 m

m ×

3.0

mm

, 5

µm);

isoc

ratic

; ace

toni

trile

:wat

er:p

erch

loric

ac

id (2

6:7

4:0

.25,

v/v

/v),

pH

2.5

with

5 M

NaO

H;

0.5

ml/

min

; 20

µl

HPL

C–U

V (3

50 n

m);

9.0

µg

/20

0 ng

for

mic

e pl

asm

a/br

ain

tissu

e; 1

5 m

in; 5

.59

min

; 150

–20,

00

0 ng

/ml i

n m

ice

plas

ma

and

100

0–2

0,0

00

ng/m

g in

bra

in t

issu

e; a

pplie

d to

a b

lood

–br

ain

barr

ier

min

ocyc

line

tran

spor

t st

udy

[31]

6Pr

otei

n pr

ecip

itatio

n w

ith a

ceto

nitr

ile

(0.2

ml,

hum

an p

lasm

a); 9

7.5%

; te

trac

yclin

e

HyP

URI

TY™

C8

(10

0 m

m ×

4.6

mm

, 5.0

µm

);

isoc

ratic

; met

hano

l:wat

er:f

orm

ic a

cid

:1.0

M

amm

oniu

m t

rifluo

roac

etic

aci

d, p

H 2

.9

(30

:70

:0.0

5:0.

1, v

/v/v

/v);

0.8

ml/

min

; 5 µ

l

LC–M

S/M

S; 8

.9 n

g; 4

.5 m

in; 2

.58

min

; 20

–40

00

ng/m

l; bi

oequ

ival

ence

stu

dy in

14

heal

thy

volu

ntee

rsPr

esen

t m

etho

d

HLB

: Hyd

roph

ilic–

lipop

hilic

bal

ance

.

Analysis of a second generation tetracycline antibiotic minocycline | Methodology

www.future-science.com 2179future science group

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540

60

1.0e52.0e53.0e54.0e55.0e56.0e57.0e58.0e59.0e51.0e62.0e63.0e64.0e65.0e66.0e67.0e68.0e69.0e61.0e72.0e73.0e74.0e75.0e76.0e77.0e78.0e7

5.0e5

1.0e6

1.5e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

6.5e6

7.5e6

8.5e6

9.0e6

9.5e6

7.0e6

8.0e6

5.5e6

3.5e6

4.5e6

2.5e6

80 100 120 140 160 180 200 220 240 260 280 300

154.2

126.2

154.1

OH OHOH

H HOH

O O

Inte

nsi

ty (

cps)

Inte

nsi

ty (

cps)

215.4 230.0 283.3 298.3

337.1

352.2

380.4395.3

423.2

441.2

OH OHOH

OHH

–NH3

–NH3

–H2O3

CONH2

CONH2

CH3N(CH3)2HO

m/z (amu)

m/z (amu)

O O

291.2269.3 321.0

337.0365.1

410.1

[M+1]+

445.2

349.2

320 340 360 380 400 420 440 460 480 500

[M+1]+

458.2

N(CH3)2N(CH3)2

Figure 1. Product ion mass spectra. (A) Minocycline (m/z 458.2 → 441.2, scan range 50–550 amu) and (B) tetracycline (internal standard, m/z 445.2 → 410.1, scan range 50–500 amu) in positive ion mode.



dissociation: 5 psi, curtain gas: 25 psi. The compound-dependent parameters, such as declustering potential, collision energy and cell exit potential were optimized at 40, 30 and 20 V for MC and 30, 25 and 15 V for IS, respectively. Quadrupole 1 and quadrupole 3 were maintained at unit resolution, while the dwell time set was 600 ms and 400 ms for MC and IS, respectively.

� Preparation of standard stock & plasma samples The MC standard stock solution of 500 µg/ml was prepared by dissolving requisite amount in methanol. This was further diluted in the same diluent to get an intermediate solution of 80.0 µg/ml. The working solutions of MC for spiking plasma calibration and quality control samples were subsequently prepared using the standard and intermediate stock solutions in methanol. The IS stock solutions of 100 µg/ml was prepared by dissolving requisite amount of TC hydrochloride in methanol. IS working solu-tion (1500 ng/ml) was prepared using the stock solution in acetonitrile. All the above solutions were stored at -70°C in amber colored bottles until use. Drug-free plasma (control [blank] plasma) was withdrawn from the deep freezer and allowed to completely thaw before use. The calibration standards (CSs) and quality control (QC) samples were prepared by spiking blank plasma with respective working solutions (5% of total volume of plasma). CSs were made at 20.0, 40.0, 75.0, 150, 250, 500, 1000, 2000, 3200 and 4000 ng/ml for MC. QCs were pre-pared at 20.0 ng/ml for LLOQ QC, 60.0 ng/ml for low quality control (LQC), 400 ng/ml for medium quality control (MQC)-2, 1200 ng/ml for MQC-1, 3000 ng/ml for high quality con-trol (HQC) and 4000 ng/ml for ULOQ QC concentrations, respectively. The spiked plasma samples at all the levels were stored at -70°C in amber colored ria vial for validation and subject sample ana lysis.

� Protocol for sample preparation Owing to the sensitive nature of MC, the entire extraction process was carried out under yellow light (570–580 nm). Prior to ana lysis, spiked plasma samples were thawed in an ice water bath and allowed to equilibrate at room temperature. The samples were adequately vortexed using a vortexer before pipetting. Aliquots of 200 µl plasma solutions containing 10 µl of working solution of MC were transferred into amber

colored polypropylene microcentrifuge vials and 400 µl working solution of IS (1500 ng/ml) was added. The vials were capped and vortexed vigorously for 1 min, followed by centrifugation at 17,949 g for 10 min at 25°C. The supernatant (50 µl) was transferred in labeled amber colored ria vials and 400 µl of dilution solution was added. The solution was thoroughly vortexed and 5 µl was used for injection in LC–MS/MS, in partial loop mode.

� Methodology for validationA thorough and complete method validation of MC in human plasma was done following the US FDA guidelines [37]. The method was validated for selectivity, sensitivity, interference check, carryover check, linearity, precision and accuracy, reinjection reproducibility, recovery, ion suppression/enhancement, matrix effect,

LOD, stability and dilution integrity. Test for selectivity was carried out in 12 dif-

ferent lots of blank human plasma including hemolyzed and lipemic plasma collected with K3-EDTA as an anticoagulant. From each of these 12 different samples, two replicates were prepared (190 µl plasma samples were spiked with 10 µl of methanol). In the first set, the blank human plasma was directly injected after extrac-tion (without analyte and IS), while the other set was spiked with only IS before extraction (total 24 samples). Furthermore, one system suitability sample at CS-2 (40.0 ng/ml) concentration and two replicates of LLOQ concentration (CS-1) were prepared by spiking 190 µl blank human plasma with 10 µl of respective working aqueous standards of MC. The blank human plasma used for spiking of one system suitability sample and LLOQ were chosen from one of these 12 samples of plasma. The acceptance criterion requires that at least 90% of selectivity samples should be free from any interference at the retention time of analyte and IS.

The interference due to commonly used medications in human volunteers was done for acetaminophen, aspirin, caffeine, cetrizine, chlorpheniramine maleate, ibuprofen and pseu-doephedrine. Their stock solutions (100 µg/ml) and working solutions (20 µg/ml) were prepared in methanol. A volume of 10 µl working solution of each commonly used medication was spiked in 190 µl of blank human plasma into amber colored polypropylene microcentrifuge vials and vortexed to mix. These plasma samples were processed without IS by the proposed extrac-tion procedure and 5.0 µl was injected to check



any possible interference at the retention time of analyte and IS. Also, the possible interference due to MC and 4-EMC at the retention time of IS was checked during the interference study. The verification of carryover of analytes was also experimentally determined. The design of the study comprised of the following sequence of injections, that is, double blank plasma (DBP) sample → two samples of LLOQ → DBP → ULOQ sample → DBP → ULOQ sample → DBP.

The linearity of the method was determined by ana lysis of seven calibration curves contain-ing ten non-zero concentrations. The area ratio response for MC/IS obtained from multiple reaction monitoring was used for regression ana-lysis. Each calibration curve was analyzed indi-vidually by using least square weighted (1/x2) linear regression, which was finalized during pre-method validation. A correlation coefficient (r) value >0.99 was desirable for all the calibration curves. The lowest standard on the calibration curve was accepted as the LLOQ, if the analyte response was at least ten-times more than that of drug-free (blank) extracted plasma.

Intra-batch and inter-batch (on three consecu-tive validation days) accuracy and precision were evaluated at six QC levels (LLOQ QC, LQC, MQC-2, MQC-1, HQC and ULOQ QC) in six replicates for MC. The precision (% coef-ficient of variation [CV]) at each concentration level from the nominal concentration should not be greater than 15%. Similarly, the mean accu-racy should be within 85–115%, except for the LLOQ where it can be from 80 to 120% of the nominal concentration. Reinjection reproduc-ibility was performed by re-injecting one vali-dation batch after an interval of 30 min. The extracts were maintained at a temperature of 25°C, while the injection samples were kept at 4°C and were injected consecutively after a gap of approximately 30 s. Also, two thirds of qual-ity control samples should meet the criteria of 85–115% of nominal concentration for accuracy and 15% CV for precision.

The relative recovery, matrix effect and pro-cess efficiency were assessed as recommended by Matuszewski et al. [38]. All three parameters were evaluated at HQC, MQC-1, MQC-2 and LQC levels in six replicates. Relative recovery (RE) was calculated by comparing the mean area response of prespiked samples (spiked before extraction) to that of extracts with post-spiked samples (spiked after extraction) at each QC level. The recovery of IS was similarly

estimated. Absolute matrix effect (ME) was assessed by comparing the mean area response of unextracted samples (spiked after extraction) with mean area of neat standard solutions (in mobile phase). The overall ‘process efficiency’ (%PE) was calculated as (ME × RE)/100. Furthermore, the effect of plasma matrix (dif-ferential matrix effect) on analyte quantifica-tion was also checked in eight different batches of K3-EDTA plasma including hemolyzed and lipemic plasma. From each batch, four samples at LQC and HQC levels was prepared (spiked after extraction) and checked for the percentage accuracy and precision (% CV). The deviation of the standards and QCs should not be more than ±15%.

Matrix ion suppression effects on the MRM LC–MS/MS sensitivity were evaluated by postcolumn analyte infusion experiment [39]. A standard solution containing 1200 ng/ml

of MC and 1500 ng/ml of IS in mobile phase was infused postcolumn via a ‘T’ connector into the mobile phase at 5.0 µl/min employing a Harvard infusion pump. Aliquots of 5.0 µl of extracted MC sample (3000 ng/ml) and a blank (without IS) sample were then injected and MRM LC–MS/MS chromatograms were acquired. Any dip in the baseline upon injec-tion of DBP would indicate ion suppression, while a peak at the retention time of MC and IS indicates ion enhancement.

All stability results were evaluated by measur-ing the area ratio response (MC/IS) of stability samples against freshly prepared comparison standards at LQC and HQC levels. Stock solu-tions of MC and IS were checked for short-term stability at room temperature and long-term sta-bility at -20°C. The solutions were considered stable if the deviation from nominal value was within ±10.0%. Bench top stability, processed sample stability at room temperature and at refrigerated temperature (4°C), freeze–thaw sta-bility and long-term stability in plasma at -70°C were performed at LQC and HQC levels using six replicates at each level. To meet the accep-tance criteria the %CV and percentage accuracy should be within ±15%. In addition, at least 2/3 QC samples should meet the criteria of ±15% of nominal concentration.

The dilution integrity experiment was per-formed with an aim to validate the dilution test to be carried out on higher MC concentrations (above ULOQ), which may be encountered during real subject samples ana lysis. A dilution integrity experiment was carried out at five times



the ULOQ concentration (20,000 ng/ml) and at HQC level for MC. Six replicate samples each of 1/10 of five times the ULOQ (2000 ng/ml) and 1/10 of HQC (300 ng/ml) concentration were prepared and their concentrations were calcu-lated, by applying a dilution factor of 10 against the freshly prepared calibration curve.

� Bioequivalence study design & statistical ana lysisThe design of the study comprised of ‘an open-label, balance, randomized, two treat-ment, two sequence, two period, single dose, crossover, comparative evaluation of relative oral bioavailability test (135 mg MC tablet of a sponsor company) and reference formulation (SOLODYN®, 135 mg MC extended release tablet from Wellspring Pharmaceutical Canada Corporation, Ontario, Canada) in 14 healthy Indian subjects under fasting conditions’. All the subjects were informed of the aim and risk involved in the study and written consent were obtained. The procedures followed while deal-ing with human subjects were based on the International Conference on Harmonization, E6 Good Clinical Practice guidelines [40]. Blood samples were collected in vacutainers containing K3-EDTA anticoagulant before (0.0 h) and at 1.0, 2.0, 2.50, 3.0, 3.25, 3.50, 3.75, 4.0, 4.25, 4.50, 5.0, 5.5, 6.0, 7.0, 8.0, 10.0, 12.0, 16.0, 24.0, 48.0, 72.0, 96.0 h of administration of drug. Blood samples were centrifuged at 1811 g at 4°C for 10 min, the plasma was separated and stored at -70°C until use. The plasma con-centration–time profiles obtained from the experimental subjects were analyzed by noncom-partmental ana lysis using WinNonlin® profes-sional software (version: 5.2.1) from Pharsight Corporation (CA, USA). An incurred sample reanalysis was also conducted by computerized random selection of subject samples (10% of total study samples analyzed) near C

max and the

elimination phase. The results obtained were compared with the data obtained earlier for the same sample using the same procedure. The percent change in the value should not be more than ±20% [41].

Results & discussion � Method development

The development of the proposed method was based on the outcome of several previous reports for determination of MC. The objective of the present work was to develop and fully validate a simple, rapid, selective and sensitive method

for MC in human plasma by LC–MS/MS for routine sample ana lysis. To achieve this aim, significant efforts were focused on the extrac-tion procedure, due to the sensitive nature of the analyte and IS, and to optimize the best possible chromatographic conditions. Also, the sensitiv-ity should be adequate enough to monitor at least five half lives of MC concentration with good accuracy and precision for subject samples.

During method development, the ionization of MC and IS was conducted in positive mode, as both the drugs are basic in nature, using electro spray ionization. The Q1 MS full scan spectra for MC and IS predominantly contained protonated precursor (M+H)+ ions at m/z 458.2 and 445.2, respectively. Furthermore, fragmen-tation was initiated using sufficient nitrogen for collision-activated dissociation and by applying 30 V collision energy to break the precursor ions. The most abundant and consistent prod-uct ions in Q3 MS spectra for MC was observed at m/z 441.2 due to the loss of a -NH

3 group,

while for IS the stable and consistent product ion was found at m/z 410.1 using 25 V colli-sion energy, which can be attributed to the loss of a water molecule and a -NH

3 group [33]. To

attain an ideal Taylor cone and a better impact on spectral response, nebulizer gas pressure was optimized at 50 psi. Fine tuning of nebulizer gas and collision activation dissociation gas was done to get a consistent and stable response. It was observed that ion spray voltage had a signifi-cant effect on the response of both the analytes and IS. At high voltage (5500 V), the response was drastically reduced and hence an optimum potential of 3500 V was kept, which gave a con-sistent and stable signal. The ion source cham-ber temperature had little effect on the signal and, hence, was maintained at 550°C. A dwell time of 600 and 400 ms was adequate and no cross talk was observed between the MRMs of analytes.

Several existing methods have critically exam-ined the best conditions for chromatographic separation of MC on C8 [21,23,28,29,31] and C18 [30,33,36] reversed-phase columns. In these meth-ods, various chromatographic parameters, such as composition and nature of mobile phase [21], buffer pH (2–7) [21,30], role of different addi-tives, such as tetra-butyl ammonium hydrogen sulfate [30], triethyl amine [33], trifluoroacetic acid [23,28], EDTA [29,30], oxalic acid [33], formic acid [36], column temperature, injection volume and flow rate [21,30,33] have been investigated for efficient separation of MC. Additives such as



tetra-butyl ammonium hydrogen sulfate and triethyl amine contribute significantly towards better selectivity, resolution and peak symme-try of TC antibiotics. EDTA helps in removing any metal ion impurities in the mobile phase through complexation and thus prevents their interaction with TCs. Chromatographic perfor-mance of TCs on polymeric styrene-divinylben-zene copolymer and C18 bonded-phase columns with several commonly used buffers, organic modifiers and counter ions has been also been discussed [42]. All these parameters were consid-ered during method development and adequate

precautions were taken to minimize any possible degradation of MC during extraction. 4-EMC and 4-ETC are the major degradation impuri-ties in MC and TC, respectively, which can be present in standard reference substances or can be formed due to degradation under pH 3.0–4.5 and light conditions. Furthermore, it was also desired that the response should be adequate with sharp peak shape and a short run time. Thus, in the present study four different col-umns were tested, namely Peerless Basic C18 (100 × 4.6 mm, 3 µm, pore size 150 Å, carbon loading 4%), HyPURITY Cyano (50 × 4.6 mm,

0.502468

101214161820222426283032343638404244

05

101520253035404550556065707580859095

0.21 0.68

0.97 1.76 2.32

3.58

3.16

4.09

1.0 1.5 2.0 2.5 3.0Time (min)

Inte

nsi

ty (

cps)

Inte

nsi

ty (

cps)

Time (min)3.5 4.0 0

2468

101214161820222426283032343638404244

0.01.0e42.0e43.0e44.0e45.0e46.0e47.0e48.0e49.0e41.0e51.1e51.2e51.3e51.4e5

1.6e51.5e5

1.7e51.8e51.9e52.0e5

0.31

1.53

1.87

2.73

2.993.61

4.40

2.67

3.18

2.54

0.90Inte

nsi

ty (

cps)

Inte

nsi

ty, c

ps

0.5 1.0 1.5 2.0 2.5 3.0Time (min)

3.5 4.0 0.5 1.0 1.5 2.0 2.5 3.0Time (min)

Tetracycline

4-Epitetracycline

3.5 4.00.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

0.0

1.0e42.0e43.0e44.0e45.0e46.0e47.0e48.0e49.0e41.0e51.1e51.2e51.3e51.4e51.5e51.6e51.7e51.8e51.9e5

3.15

2.58

2.68

3.18

Inte

nsi

ty (

cps)

Inte

nsi

ty (

cps)

0.50

200400600800

1000120014001600180020002200240026002800300032003400360038004000

1.0 1.5 2.0 2.5 3.0Time (min)

3.5 4.0 0.5 1.0 1.5 2.0 2.5 3.0Time (min)

3.5 4.0

TetracyclineMinocycline

4-Epiminocycline4-Epitetracycline

0.0

1.0e42.0e4

3.0e4

4.0e4

5.0e46.0e47.0e4

8.0e4

9.0e41.0e5

1.1e5

1.2e51.3e5

1.4e5

0.01.0e42.0e43.0e44.0e45.0e46.0e47.0e48.0e49.0e41.0e51.1e51.2e51.3e51.4e51.5e51.6e51.7e51.8e51.9e52.59 3.19

2.683.16

Inte

nsi

ty (

cps)

Inte

nsi

ty (

cps)

0.5 1.0 1.5 2.0 2.5 3.0Time (min)

3.5 4.0 0.5 1.0 1.5 2.0 2.5 3.0Time (min)

3.5 4.0


4-Epitetracycline4-Epitetracycline

Figure 2. MRM ion chromatograms of minocycline (m/z 458.2 → 441.2) and tetracycline (IS, m/z 445.2 → 410.1). (A) Double blank plasma (without analyte and IS); (B) blank plasma with IS; (C) minocycline at LLOQ and IS; and (D) real subject sample at Cmax after administration of 135 mg dose of minocycline.



5 µm, pore size 190 Å, carbon loading 4%), BetaBasic CN (100 × 2.1 mm, 5 µm, pore size 150 Å, carbon loading 5%) and HyPURITY C8 (100 × 4.6 mm, 5 µm, pore size 190 Å, carbon loading 8%). These columns have demonstrated superior performance for separation of basic compounds with good peak shapes as they are base deactivated. However, as they have differ-ent carbon loading, pore size and surface area, their retention ability and separation efficiency for MC and TC was investigated. Different volume ratios (60:40, 70:30, 80:20 and 90:10, v/v) of water-methanol and water-acetonitrile

combinations were tried as the mobile phase, along with formic acid (0.01–0.05%), ATFA (1–5 M) and ammonium acetate buffers (2–20 mM). In addition, the effect of flow rate (0.5–1.0 ml/min), column temperature (30–40°C) and split ratio, ion source:waste (20:80, 30:70, 40:60 v/v) was also studied, which were responsible for acceptable chromatographic peak shapes. The run time on Peerless basic C18 col-umn was relatively long (greater than 5 min) and the response was not reproducible for the proposed linear range. Although separation was possible on the other three columns with

0.502468

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101520253035404550556065707580859095

0.21 0.68

0.97 1.76 2.32

3.58

3.16

4.09

1.0 1.5 2.0 2.5 3.0Time (min)

Inte

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ty (

cps)

Inte

nsi

ty (

cps)

Time (min)3.5 4.0 0

2468

101214161820222426283032343638404244

0.01.0e42.0e43.0e44.0e45.0e46.0e47.0e48.0e49.0e41.0e51.1e51.2e51.3e51.4e5

1.6e51.5e5

1.7e51.8e51.9e52.0e5

0.31

1.53

1.87

2.73

2.993.61

4.40

2.67

3.18

2.54

0.90Inte

nsi

ty (

cps)

Inte

nsi

ty, c

ps

0.5 1.0 1.5 2.0 2.5 3.0Time (min)

3.5 4.0 0.5 1.0 1.5 2.0 2.5 3.0Time (min)

Tetracycline

4-Epitetracycline

3.5 4.00.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

0.0

1.0e42.0e43.0e44.0e45.0e46.0e47.0e48.0e49.0e41.0e51.1e51.2e51.3e51.4e51.5e51.6e51.7e51.8e51.9e5

3.15

2.58

2.68

3.18

Inte

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cps)

Inte

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cps)

0.50

200400600800

1000120014001600180020002200240026002800300032003400360038004000

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3.5 4.0 0.5 1.0 1.5 2.0 2.5 3.0Time (min)

3.5 4.0


4-Epiminocycline4-Epitetracycline

0.0

1.0e42.0e4

3.0e4

4.0e4

5.0e46.0e47.0e4

8.0e4

9.0e41.0e5

1.1e5

1.2e51.3e5

1.4e5

0.01.0e42.0e43.0e44.0e45.0e46.0e47.0e48.0e49.0e41.0e51.1e51.2e51.3e51.4e51.5e51.6e51.7e51.8e51.9e52.59 3.19

2.683.16

Inte

nsi

ty (

cps)

Inte

nsi

ty (

cps)

0.5 1.0 1.5 2.0 2.5 3.0Time (min)

3.5 4.0 0.5 1.0 1.5 2.0 2.5 3.0Time (min)

3.5 4.0


4-Epitetracycline4-Epitetracycline

Figure 2. MRM ion chromatograms of minocycline (m/z 458.2 → 441.2) and tetracycline (IS, m/z 445.2 → 410.1). (A) Double blank plasma (without analyte and IS); (B) blank plasma with IS; (C) minocycline at LLOQ and IS; and (D) real subject sample at Cmax after administration of 135 mg dose of minocycline.



better peak shape and a reproducible response, the retention was inadequate on HyPURITY Cyano and Betabasic CN (less than 2.0 min). This could possibly be due to low carbon load-ing (4–5%) compared with HyPURITY C8 column (8%).

The use of HyPURITY C8 chromatography column helped in the separation and adequate retention of both the analytes in a short time with good peak shape. The retention time for MC and IS was 2.58 and 3.18 min, respectively, at an optimum flow rate of 0.8 ml/min. The rep-resentative chromatograms of extracted blank plasma (without IS and analyte), blank plasma fortified with IS, MC at LLOQ and a real sub-ject sample chromatogram for MC at C

max is

shown in Figure 2a–D. The capacity factors, which describe the rate at which the analytes

migrate through the column, were 1.35 and 1.89 for MC and TC, respectively, based on the dead time of 1.1 min. The selectivity fac-tor (α) of the column for their chromatographic separation was 1.4. The number of theoreti-cal plates obtained for MC and TC were 526 and 647, respectively, with a resolution factor of 1.3 between the two drugs. Moreover, the peaks observed at 3.15 min and 2.68 min, cor-responding to 4-EMC and 4-ETC (confirmed with reference standard) were chromatographi-cally separated from their parent drugs with a resolution factor greater than 1.5. However, no efforts were made to quantify these impuri-ties. The likely source of these impurities could be their presence in the reference standards of the analyte and IS, as their intensity was con-sistent at all CSs, QCs and subject samples. A

260024002200200018001600140012001000

800600400200

0 0.01.0e42.0e43.0e44.0e45.0e46.0e46.0e48.0e49.0e41.0e51.1e51.2e51.3e5

1.0 1.5 2.0 2.5 3.0

2.53

3.16 2.69

3.19

Time (min)

Inte

nsi

ty (

cps)

Inte

nsi

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cps)

Minocycline

4-Epiminocycline

4-Epitetracycline

Tetracycline

Time (min)

3.5 4.00.5 1.0 1.5 2.0 2.5 3.0 3.5 4.00.5

Figure 3. MRM ion chromatograms of (A) minocycline (m/z 458.2 → 441.2) and (B) tetracycline (IS, m/z 445.2 → 410.1) at LLOQ level for a sample processed under natural light.

Table 2. Intra- & inter-batch accuracy and precision for minocycline.

Quality control ID

Concentration added(ng/ml)

Intra-batch Inter-batch

Observations (n)

Mean concentration found (ng/ml)†

Accuracy (%)

CV (%)

Observations (n)

Mean concentration found (ng/ml)‡

Accuracy (%)

CV(%)

LLOQ 20.0 6 19.5 97.6 4.9 18 19.5 97.6 3.7

LQC 60.0 6 61.7 102.8 6.8 18 60.9 101.4 7.9

MQC-2 400 6 426 106.5 3.0 18 418 104.4 6.0

MQC-1 1200 6 1250 104.2 2.1 18 1242 103.5 6.7

HQC 3000 6 3186 106.2 4.8 18 3103 103.4 6.4

ULOQ 4000 6 4047 101.2 4.4 18 3939 98.5 5.2

†Mean of six replicate observations at each concentration.‡Mean of 18 replicate observations over three different analytical runs.HQC: High-quality control; LQC: Low-quality control; MQC: Medium-quality control.



mobile phase consisting of 0.05% formic acid and 0.01%, 1 M ATFA solution in deionized water:methanol (70:30, v/v) and having pH 2.9 was found most appropriate for faster elu-tion, improved efficiency and peak shape. It was observed that ATFA helped in better retention of the analyte and IS with the stationary phase and thus contributed towards improved selec-tivity, while 0.05% formic acid was adequate to achieve sufficient resolution and good peak shape. At flow rates higher than 0.8 ml/min there was more tailing of the peaks, while for lower flow rates the run time increased, which gave relatively poor resolution. The column oven temperature of 30°C helped in achieving good separation at the same time maintaining column stability. The maximum on-column loading of MC at ULOQ was 8.9 ng per sample injection volume. Also, the reproducibility of retention times for MC, expressed as %CV was ≤1% for 100 injections on the same column.

Ideally, a deuterated analogue should be the first-choice internal standard, but due to its unavailability, TC belonging to the same class of antibiotics was used as an IS to minimize ana-lytical variation due to solvent dilution, integrity of the column and ionization efficiency. TC had similar chromatographic behavior and was easily separated from the analyte. There was no effect of IS on analyte recovery, sensitivity or ion sup-pression. Also, the validation results obtained from this LC–MS/MS methodology encouraged its selection as an IS for the present study.

The majority of the procedures have used solid-phase extraction for quantitative extrac-tion of MC and other TCs from biological matrices [28,29,31], environmental samples [36]

and honey samples [33]. Mascher [27] and Araujo et al. [35] have employed LLE with ethyl acetate and diethyl ether/dichloromethane with mean recoveries of 76.4 and 86.2%, respectively. So far there are no reports on the use of protein pre-cipitation of analyte from any biological matrix, except urine [34]. In this procedure, several vet-erinary drugs were screened by directly diluting the untreated urine 1:10 (v/v) with 5% aceto-nitrile into well plates. In our effort to develop a simple, quick and economical procedure for sample preparation with minimum work-up (fewer processing steps), protein precipitation was initiated with ethanol, methanol, acetone and acetonitrile solvents. The IS solution was made in the precipitating solvents to reduce the number of steps during sample processing. Owing to the sensitive nature of TCs, care was taken to prevent direct exposure of the drugs to

Table 3. Absolute matrix effect, relative recovery and process efficiency for minocycline.

QC level

A†

(%CV)B‡ (%CV)

C§ (%CV)

Absolute matrix effect (%ME)¶

Relative recovery (%RE)#

Process efficiency (%PE)††

LQC 0.065(5.6)

0.064(6.3)

0.063(1.9)

98.4 98.3 96.7

MQC-2 0.449(3.3)

0.435(3.8)

0.427(1.6)

96.9 98.1 95.1

MQC-1 1.392(1.2)

1.310 (1.1)

1.265(4.3)

94.1 96.6 90.9

HQC 3.527(1.5)

3.253(1.5)

3.156(2.4)

92.2 97.0 89.4

†Mean area ratio response of six replicate samples prepared in mobile phase (neat samples).‡Mean area ratio response of six replicate samples prepared by spiking in extracted blank plasma. §Mean area ratio response of six replicate samples prepared by spiking before extraction.¶(B/A) × 100#(C/B) × 100††(C/A) × 100 = (ME × RE)/100CV: Coefficient of variation; HQC: High-quality control; LQC: Low-quality control; MQC: Medium-quality control.

Table 4. Differential matrix effect in different lots of human plasma at low quality control and high quality control levels for minocycline (n = 4).

Plasma lots LQC (60.0 ng/ml) HQC (3000 ng/ml)

Mean calculated concentration† (%CV)

Mean calculated concentration† (%CV)

Lot-1 57.04 (4.0) 2965 (5.0)Lot-2 56.84 (2.5) 2912 (3.2)Lot-3 56.40 (2.2) 2938 (4.5)Lot-4 55.73 (3.2) 2953 (3.0)Lot-5 56.13 (2.0) 2921 (3.6)Lot-6 55.08 (2.8) 2904 (2.7)Lot-7 (hemolyzed) 57.58 (2.8) 2948 (5.3)Lot-8 (lipemic) 56.63 (3.7) 2937 (1.8)†Mean of four replicate observations at each concentration.CV: Coefficient of variation; HQC: High-quality control; LQC: Low-quality control; n: Number of replicates at each level.



natural light; hence, the entire procedure was carried out under yellow light (570–580 nm). The extracts obtained with ethanol, methanol and acetone were clear but the recovery was in the range of 60–70%. However, quantitative and precise recoveries (>96%) were obtained for MC and TC using acetonitrile as the precipi-tating agent compared with other solvents with negligible ion suppression. Significant reduction in recovery (<35%) was observed for both the drugs in the presence of natural light, which was not consistent at all QC levels. Figure 3 shows considerable conversion of MC and TC to their corresponding epimers, 4-EMC and 4-ETC, respectively for the sample, processed under natural light.

� Assay performance & validation results During the entire method validation, the precision (% CV) of system suitability test was observed in the range of 0.07–0.30% for

the retention time and 1.5–1.9% for the area response of MC and IS. Carry-over evalua-tion was performed to ensure that it does not affect the accuracy and precision of the pro-posed method. Almost negligible area (less than 1% of LLOQ area) was observed in DBP run after ULOQ, which suggests no carry-over of the analyte in subsequent runs. Moreover, no ghost peaks appear during the ana lysis of blank samples.

The aim of performing selectivity check with 12 different plasma samples was to determine the extent to which endogenous plasma com-ponents might contribute to the interference at the retention time of analyte and the IS and, thus, ensure the authenticity of the results for study sample ana lysis. All samples studied were found free from any endogenous interference. Representative chromatograms in Figure 2a & b of extracted blank plasma (without IS and ana-lyte), blank plasma fortified with IS demonstrate

0.5 1.0 1.5 2.0

0.03

0.25 0.40 0.59 0.720.98

0.74

XIC of +MRM (2 pairs): 458.2/441.2 amu from Sample 1 (BLK/BLK) of Ion Suppression_Enhancement

XIC of +MRM (2 pairs): 445.2/410.2 amu from Sample 1 (BLK/BLK) of Ion Suppression_Enhancement

0.93 1.26 1.39 1.54 1.79 4.19

Max. 4.5e4 cps.

Max. 6.6e4 cps.

4.38

TIme (min)

TIme (min)

Inte

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ty (

cps)

Inte

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2.5 3.0 3.5 4.0

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1.5e5

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1.0e5

2.0e5

4.0e5

3.0e5

5.0e5

5.8e5

1.0 1.5 2.0 2.5 3.0 3.5 4.0

1.16 1.391.53 1.75 2.28 2.37 3.63 4.003.94

Figure 4. Representative postcolumn analyte infusion MRM LC–MS/MS overlaid chromatograms for minocycline and tetracycline. (A) XIC chromatogram of minocycline (m/z 458.2 → 441.2) (B) XIC of tetracycline (IS, m/z 445.2 → 410.1).XIC: Exact ion current.



the selectivity of the method. None of the com-monly used medications by subjects showed interfering signals at the retention time of MC or IS.

All six calibration curves were linear over the concentration range of 20.00–4000 ng/ml with correlation coefficient r > 0.9981. A straight line fit was made through the data points by least square regression ana lysis to give the mean linear equation:

y = (0.0008 ± 0.00009)x + (0.0051 ± 0.0001)

where y is the peak area ratio of the analyte/IS and × the concentration of the analyte. The accuracy and precision (%CV) observed for

the calibration curve standards ranged from 95.5 to 107.8% and 1.6 to 5.2%, respectively. The LLOQ in the standard curve that can be measured with acceptable accuracy and pre-cision was found to be 20.0 ng/ml at a S:N ratio of ≥90 with a LOD of 0.50 ng/ml. The sensitivity could have been brought down to less than 3 ng/ml; however, it was not required in the present work based on subject sample results. This would have increased the linear range 1333-fold compared with 200-fold in the proposed method.

The intra-batch and inter-batch precision and accuracy were established from validation runs performed at LLOQ QC, LQC, MQC-2, MQC-1, HQC and ULOQ QC levels (Table 2).

Table 5. Stability results for minocycline under different conditions (n = 6).

Stability Storage Condition Level A† (ng/ml) % CV % change‡

Bench top stability In ice water bath at room temperature (25 h)

LQC 60.1 2.4 0.1HQC 3019 2.6 0.6

Processed sample stability (extracted samples)

Auto sampler(4°C, 72 h)

LQC 60.2 3.4 0.3HQC 3221 6.5 7.3

Processed sample stability (extracted samples)

Room temperature (48h) LQC 60.5 8.7 0.8HQC 2893 2.9 -3.6

Freeze–thaw stability After 6th cycle at -70°C LQC 59.7 2.9 -0.4HQC 3226 1.5 7.5

Long term stability 99 days at -70°C LQC 63.1 1.4 5.2HQC 3258 2.0 8.6

†mean stability sample

‡%Re

changeMean of initial and repeat values

peat value Initial value100#=

-c m

CV: coefficient of variance; n: number of replicates at each level.

0

Pla

sma

con

cen

trat

ion

(n

g\m

l)

10

100

1000

10000

8 16 24 32 40 48

Time (h)

Reference

56 64 72 80

Test

88 96

Figure 5. Mean plasma concentration–time profile of minocycline after oral administration of 135 mg test and reference formulation to 14 healthy subjects under fasting conditions.



The intra-batch precision (%CV) ranged from 2.1 to 6.8 and the accuracy was within 97.6 to 106.5%. For the inter-batch experiments, the precision varied from 3.7 to 7.9 and the accuracy was within 97.6 to 104.4%.

Matuszewski et al. have described approaches for studying, identifying and eliminating the effect of matrix on the results of quantitative analyses by LC–MS/MS [38]. For quantitative assessment of matrix effect, they introduced the concept of ‘absolute’ versus ‘differential’ matrix effect. In addition, detailed experiments were described to calculate the true recovery of ana-lytes using HPLC–MS/MS, eliminating the uncertainty about the effect of matrix on quan-tification. Based on their approach, the terms peak area ratio (analyte/IS) response obtained in neat solution/mobile phase (A), peak area ratio (analyte/IS) response for standards spiked in extracted blank plasma (B) and peak area ratio (analyte/IS) response for standards spiked before extraction (C) have been calculated at LQC, MQC-2, MQC-1 and HQC levels as shown in Table 3. To account for ion suppres-sion and enhancement, the ratio B/A × 100 gives the measure of absolute matrix effect (%ME). The relative or true recovery (%RE)

is defined as C/B × 100 that is unaffected by the matrix. The value C/A × 100 is expressed as process efficiency or absolute recovery, which does not account for the matrix effect and can also be calculated by multiplying %ME with %RE. The mean process efficiency obtained for MC was between 89.4 and 96.7% at all QC levels, while the recovery of IS was 96.1% with %CV of 3.5. The more important parameter in the evaluation and validation of a bioana-lytical method in biofluids is the demonstra-tion of the absence of a ‘differential or relative’ matrix effect, which compares matrix effect values between different lots of blank plasma. This is expressed in terms of precision (%CV) in replicate measurements as shown in Table 4. For different lots of plasma samples, the varia-tion in %CV was within 1.8–5.3% at LQC and HQC levels.

The average matrix factor value calculated as the response of post-spiked sample/response of neat solutions in mobile phase at the LLOQ levels was 0.99, which indicates a minor sup-pression of 2%. Furthermore, results of a post-column infusion experiment in Figure 4 indi-cate no ion suppression or enhancement at the retention time of MC or IS. There was a minor

Table 6. Mean pharmacokinetic parameters of minocycline after oral administration of 135 mg minocycline tablet formulation in 14 healthy Indian subjects under fasting condition.

Parameter Reference Test

Mean ± SD Mean ± SDCmax (ng/ml) 994 ± 136.9 1123 ± 180.9

Tmax (h) 3.66 ± 0.48 3.77 ± 0.80

t1/2 (h) 17.70 ± 2.68 19.01 ± 3.91

AUC0–96 h (h.ng/ml) 19,966 ± 4087 21,046 ± 4277

AUC0–inf (h.ng/ml) 20,816 ± 2241 22,122 ± 2359

Kel (1/h) 0.040 ± 0.007 0.038 ± 0.009

Cmax: Maximum plasma concentration.Tmax: Time point of maximum plasma concentration.t1/2: Half life of drug elimination during the terminal phase.AUC0–96 h: Area under the plasma concentration–time curve from 0 h to 96 h.AUC0–inf: Area under the plasma concentration–time curve from 0 h to infinity.

Table 7. Comparison of treatment ratios and 90% CIs of natural log(Ln) - transformed parameters for test and reference formulations of minocycline in 14 healthy Indian subjects.

Parameter Ratio (test/reference; %)

90% CI(lower–upper)

Power Intra-subject variation (%CV)

Ln Cmax (ng/ml) 112.9 107.2–117.7 0.9666 10.4

Ln AUC0–96 h (h.ng/ml) 105.7 98.0–111.9 0.9991 8.5

Ln AUC0-inf (h.ng/ml) 106.7 99.3–112.6 0.9994 8.0



suppression near the dead time between 1.2 and 1.5 min; however, that did not affect the quantitation of MC.

Stability experiments were performed to eval-uate the analyte stability in stock solutions and in plasma samples under different conditions, simulating the same conditions that occurred during study sample ana lysis. The stock solution of MC was stable at room temperature for 6 h and at -20°C for 31 days. The intermediate stock solution of MC in methanol was stable at room temperature for 29 h and at -20°C for 16 days with percentage change of 2.5 and 0.3%, respec-tively. MC was found stable in controlled plasma (in an ice water bath) at room temperature up to 25 h and for six freeze–thaw cycles. The analyte in extracted plasma samples were stable for 72 h under refrigerated conditions (4°C) and for 48 h under room temperature. The spiked plasma samples of MC stored at -70°C for long-term stability were found stable for minimum period of 99 days. The values for the percent change for all the stability experiments are compiled in Table 5.

The precision values for dilution integ-rity of 1/10 of 5 × ULOQ (2000 ng/ml) and 1/10 of HQC (300 ng/ml) concentration were 1.3 and 3.4%, while the accuracy results were within 103.4 and 107.1% respectively, which is within the acceptance limit of 15% for precision (% CV) and 85 to 115% for accuracy.

� Application of the method on healthy human subjectsThe validated method was applied to a pharmaco kinetic study of MC in 14 healthy Indian adult male subjects who received 135 mg test and reference formulations under fasting conditions. Figure 5 shows the plasma concentration of MC versus time profile in human subjects under fasting conditions. The method was sensitive enough to monitor their plasma concentration up to 96.0 h. In total, approximately 656 samples, including the cali-bration, QC and volunteer samples were run and analyzed successfully. The precision and accuracy for calibration and QC samples were well within the acceptable limits. The mean pharmacokinetic parameters obtained for the test and reference formulation are presented in Table 6. The equivalence statistics of bio-availability for the pharmacokinetic param-eters of the two formulations are summarized in Table 7. No statistically significant differ-ences were found between the two formulations

in any parameter. The mean log-transformed ratios of the parameters and their 90% CIs were all within the defined bioequivalence range. The percentage change in the randomly selected subject samples for incurred samples (assay reproducibility) ana lysis was within ±9.0% (Table 8). This authenticates the repro-ducibility and ruggedness of the proposed method. Furthermore, there was no adverse event during the course of the study.

� Comparison with reported methods The method presented has the highest sensi-tivity compared with all other methods [27–29,31] developed in plasma for MC except one report [35], which has the LLOQ of 5 ng/ml. The method employs a simple and straightfor-ward protein precipitation for plasma sample preparation with acetonitrile using a minimum volume of human plasma (200 µl) compared with existing methods. The organic solvent consumption per sample ana lysis (extraction

Table 8. Incurred sample reanalysis data for minocycline.

Sr. No. Original concentration(ng/ml)

Repeat concentration(ng/ml)

Mean % difference

1 1284 1302 1293 1.39

2 137 143 140 4.15

3 782 760 771 -2.96

4 73.5 72.0 72.7 -2.06

5 1187 1209 1198 1.84

6 117 120 118.5 3.04

7 1139 1119 1129 -1.77

8 64.8 60.5 62.6 -6.75

9 1075 1053 1064 -2.07

10 72.3 66.2 69.3 -8.76

11 1200 1179 1190 -1.77

12 140 146 143 3.99

13 925 938 931.5 1.41

14 133 130 131.5 -2.97

15 899 888 893.5 -1.24

16 175 180 177.5 2.93

17 1162 1148 1155 -1.21

18 76.3 72.4 74.4 -5.27

19 446 453 449.5 1.47

20 78.1 72.9 75.5 -6.84

21 837 802 819.5 -4.33

22 93.4 87.9 90.6 -5.98



and chromatography) was only 1.7 ml, which is considerably lower compared with all other methods. Ion suppression/enhancement was checked by postcolumn infusion technique and the quantitative assessment of matrix effect was evaluated by calculating the abso-lute and differential matrix effect, which is not reported in previous studies. Similarly, incurred sample reana lysis, which has now become mandatory for clinical and nonclini-cal studies has been demonstrated to prove the reproducibility of the proposed method in healthy subject samples. Table 9 illustrates the potential advantage of the present method over reported procedures.

ConclusionThe objective of this work was to develop a quick, selective, sensitive, rugged and a high-throughput method for estimation of MC in human plasma, especially to meet the require-ment for routine subject sample ana lysis. The protein precipitation extraction using acetoni-trile employed in the present work gave consis-tent and quantitative recovery for MC. The run time per sample ana lysis of 4.5 min suggests high throughput of the proposed method. The maximum on-column loading at ULOQ was 8.9 ng for 5.0 µl injection volume, which helps in maintaining the efficiency and the lifetime of the column. Moreover, the LOQ is low enough to monitor at least five half lives of MC concentra-tion with good intra- and inter-assay reproduc-ibility (%CV) for the QCs. The sensitivity of the proposed method is adequate to support a wide range of pharmacokinetic/bioequivalence studies. In addition, incurred sample reana lysis is demonstrated to ensure the reproducibility of study data.

Future perspectiveAs evident from literature, very few methods are reported for the estimation of MC in biological matrices by LC–MS/MS, with a limit of quan-titation down to 5 ng/ml. Moreover, not much work has been done during the last 5–6 years on the quantitation of this drug. However, with the rapid advancement in LC–MS/MS instru-mentation it would be possible to have much more sensitive and rapid bioanalytical assays following standard guidelines. Furthermore, the quantitation of metabolites along with MC shall be of interest to the researchers working in the area of bioanalytical method development and validation. Ta

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Executive summary

� A simple, selective and sensitive method is proposed for the determination of minocycline in human plasma by LC–MS/MS.

� Plasma samples were prepared by protein precipitation with acetonitrile using 200 µl human plasma.

� Under the optimized isocratic conditions, it was possible to separate chromatographically the impurities of 4-EMC and 4-ETC present in minocycline and tetracycline (IS) respectively.

� The proposed method was fully validated as per US FDA guidelines for its selectivity, interference check, sensitivity, carryover check, linearity, precision and accuracy, reinjection reproducibility, relative and absolute recovery, differential and absolute matrix effect, ion suppression/enhancement, stability and dilution integrity.

� The proposed method is rugged, rapid (4.5 min) and adequately sensitive (20.0 ng/ml) for routine subject sample ana lysis.

� The reproducibility of the study data has been demonstrated through incurred sample reana lysis test.

AcknowledgementsThe authors are indebted to Vijay Patel (BA Research India Ltd, Ahmedabad) for providing nec-essary facilities to carry out this work. We gratefully acknowledge Anshul Dogra, Arpana Prasad (BA Research India Ltd) for their continuous support, motivation and assistance during the course of this project.

Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the sub-ject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert t estimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research The authors state that they have obtained appropri-ate insti tutional review board approval or have fol-lowed the princi ples outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investi gations involv-ing human subjects, informed consent has been obtained from the participants involved.

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Analysis of a second-generation tetracycline antibiotic minocycline in human plasma by LC–MS/MS