Penicillin V

7/25/2019 Penicillin V

1/11

SAMPLE

Matrix:

blood

Sample preparat ion:

Condition a Baker C18 SPE cartridge with 5 mL water and 5 mL

2

NaCl, do not allow to run dry. 2 mL Plasma + 30 mL water + 2 mL 170 mM sulfuric

acid H- 2 mL 5 sodium t un gs ta te solution, vortex for 30 s, centrifuge at 2200 g for 10

m in, filter sup er na ta nt (GF/B glas s fiber filter), add 10 mL 20 NaC L, mix, add to th e

SPE cartridge at 3 mL/min, wash with 5 mL 2 NaCl, wash with 5 mL water, draw air

thro ugh cartridge for 5 min, elu te with 500 JJLLelution solution. Add 500 |xL de rivatiz ation

reagent to the eluate, vortex for 20 s, allow to react at 65 for 30 min, cool to room

tem pe ra tu re , vortex, filter (0.45 jxm), inject 50-100 jxL aliqu ots. (Pr epa re der ivatiza tion

reag ent by dissolving 34.45 g

1 2 4-triazole

in 150 mL water, add 25 mL 10 mM mercuric

chloride solution, mix, adjust t he pH to 9.0 0.5 with 5 M NaO H, dilute to 250 mL with

water. Prepare elution solution by mixing 60 mL MeCN and 5 mL buffer and making up

to 100 mL with water. The buffer was 0.994 g Na

2

HPO

4

+ 1.794 g NaH

2

PO

4

.H

2

O in 100

mL water, pH 6.5.)

HPLCVARIABLES

Column: 150 X 3.9 4 |xm Nova-Pak C18

Mobile phase:

MeCN-.buffer 2 5:7 5 (Buffer con tained 4.969 g Na

2

HPO

4

+

8.969

g

NaH

2

PO

4

-H

2

O + 2.482 g anhydrous sodium thiosulfate per liter.)

Flow rate: 1

Injection volume:

50-100

Detector: UV325

CHROMATOGRAM

Retention time: 5.8

Internal standard:penicillin V

OTHER SUBSTANCES

Extracted: penicillin G

KEY WORDS

plasma; cow; SPE; penicillin V is IS

REFERENCE

Boison, J.O.; K orsrud, G.O.; MacNeil, J.D.; Keng, L.; Papich, M. D eterm ination of penicillin G in bovine

plasma by high-performance liquid chromatography after pre-column derivatization. J.Chromatogr.,

1992,

576, 315-320

SAMPLE

Matrix: blood


500 |xL Serum + 100 |xL 50 fxg/mL chloramphenieol in water + 1

mL 1 M pH 3.0 sodium acetate + 5 mL diethyl ether, sh ake for 20 min, centrifuge at

1200 g. Remove the organic layer and evaporate it to dryness at 40 under a stream of

nitrogen. Reconstitute the residue in 100 jxL water, inject a 50 jxL aliquot.

Penicillin V

Molecular formu la: C

16

H

18

N

2

O

5

S

Molecular weight:

350.4

CAS Reg istry No :

87-08-1,

132-98-9 potassium salt),

5928-8 4-7 benzathine), 63690-57-3 benzathine

tetrahydrate), 6591-7 2-6 hydrabamine)


2/11

HPLC VARIABLES

Column:

300 X 3.9 10 |xm ixBondapak C18

Mobile phase: M eCN : 10 mM pota ssium aceta te buffer 20 :80 , pH 6.5

Fl ow rate: 1.6

Inject ion volum e: 50

De tector: UV 215

CHROMATOGRAM

Retent ion t ime: 3 .10

Internal s tandard:

chloramphenicol (5.10)

Limit of detect ion:

30 ng/mL

OTHER SUBSTANCES

Noninterfer ing:

amikacin, amiloride, amoxicillin, ampicillin, cephalexin, doxycycline,

ethosuximide, gentamicin, hydrochlorothiazide, netilmicin, phenacetin, phenemal, phen-

ytoin, primidone, sisomicin, tetracycline, tobramycin

Interfering:

cloxacillin, penicillin G procaine

KEYWORDS

serum

REFERENCE

Lindberg, R.L.; Huupponen, R.K.; Huovinen, P. Rapid high-pressure liquid chromatographic method for

analysis of phenoxymethylpenicillin in human serum.

Antimicrob.Agents Chemother.,

1984,

26,

300-302

SAMPLE

Matrix: blood, urine


Pl asm a. 1 mL Pla sm a + 50 u,L 100 |xg/mL oxacillin in w ate r + 20

|JLL

4 aque ous sodium dodecyl hydro gen sulfate solution, shak e for 30 m in, filter (Amicon

MPS-I micropartition system, YMT membrane) while centrifuging, adjust the pH of the

ultrafiltrate to 6.3-6.5 with pH 4 citrate buffer, inject a 500 |xL aliquot onto column A

with mobile ph ase A and e lute to was te, after 10 min elute th e con tents of column A onto

column B with mobile pha se B , elute w ith mobile ph ase B , mon itor the effluent from

column B. Urine. 5-100 JJLLU rine + 50 |xL 100 jxg/mL oxacillin in wa ter, m ak e up to 500

IxL with w ater, inject on to column A wit h m obile ph ase A an d e lute to w ast e, after 10

min elute th e co ntents of column A onto column B w ith m obile pha se B , elute w ith mobile

phase B, monitor the effluent from column B.

HPLCVARIABLES

Column:

A 50 X 4 Nucleosil 5-C18; B 250 X 5 Nucleosil 5-C18

Mobile phase:

A MeCN: 33 mM N aH

2

PO

4

5:95; B M eCN: 33 mM N aH

2

PO

4

20:80

Inject ion vo lum e: 500

Detector: UV210

CHROMATOGRAM

Internal s tandard:

oxacillin

Limit of quant i tat ion: 50 ng/mL

KEYWORDS

plasma; column-switching; pharmacokinetics; ultrafiltrate

REFERENCE

Lintz, W.; Hirsch, L; Osterloh, G.; Schmidt-Bothelt, E.; Sous, H. Bioverfugbarkeit von Penicillin V in

einer wa(3rigen Zubere itungsform [Bioava ilability of penicillin V in aque ous dosage forms].Arzneim-

ittelforschung, 1984,34, 6 6 - 7 1


3/11

SAMPLE

Matrix:

cheese, milk, yogurt

Sample preparat ion: Condition a 6 mL 500 mg Bond Elut C 18 SPE c artridg e with 20 mL

MeO H, 20 mL water, a nd 10 mL 2 NaC l, do not allow to go dry. 5 mL Milk (or 5 g

yogurt or cottage cheese + 4 mL 1 M pH 6 pho sph ate buffer) + 25 mL wate r + 4 mL 170

mM sulfuric acid + 40 mL 5 sodium tu ng st at e, vortex for 30 s, centrifuge a t 1500 g for

10 min, remove the s up ern ata nt, add 10 mL 20 NaC l to the residu e, vortex for 10 s,

centrifuge. Combine the supernatants and add them to the SPE cartridge, wash with 10

mL 2 NaC l, was h with 10 mL water, elute with 1 mL MeCN : 200 mM pH 6.5 sodium

phosphate buffer:water 60 :5 :35 . Add 1 mL reagent to the eluate, vortex for 10 s, hea t

at 65 for 30 min, cool to room temperature, vortex, filter (Aero 0.45 |xm), inject a 50-100

jxL aliquot of the ni tra te. (Prep are reag ent by dissolving 34.45 g1 2 4-triazolein 150 mL

water, add 25 mL 10 mM mercuric chloride solution, mix, adjust pH to 9.0 0.5 with 5

M NaOH, make up to 250 mL with water.)

HPLCVARIABLES

Column:

150 X 3.9 4 jim Nova-Pak C18

Mobile phase: M eCN : buffer 2 5: 75 (Buffer w as 4.696 g Na

2

HPO

4

, 8.969 g NaH

2

PO

4

-H

2

O,

and 2.482 g anhydrous sodium thiosulfate in 1 L water.)

Flow rate: 0 .8

Inject ion volume: 50-100

Detector: UV

32 5

CHROMATOGRAM

Retent ion t ime: 7

Internal s tandard:

penicillin V

OTHER SUBSTANCES


KEYWORDS

derivatization; cow; SPE; penicillin V is IS

REFERENCE

Boison, J.O.K.; Keng, L.J.-Y; MacNeil, J.D. Analysis of penicillin G in milk by liquid chromatography.

J.AOAC Int. 1994 77 565-570

SAMPLE

Matrix: cheese, milk, yogurt

Sample preparat ion: Condition a 6 mL 500 m g Bond Elut C 18 SPE c artridge w ith 20 mL

MeOH, 20 mL water, and 10 mL 2 NaCl, do not allow to go dry. 5 mL Milk (or 5 g

yogu rt or cottage cheese + 4 mL 1 M pH 6 pho sph ate buffer) + 25 mL water + 4 mL 170

mM sulfuric acid + 40 mL 5 sodium tu ng st ate , vortex for 30 s, centrifuge a t 1500 g for

10 min, remove the su pe rn ata nt, add 10 mL 20 NaC l to the residu e, vortex for 10 s,

centrifuge. Combine the supernatants and add them to the SPE cartridge, wash with 10

mL 2 NaCl, wa sh with 10 mL water, elute with 750

JJLL

MeCN : 200 mM ammonium

ac eta te: w ate r 6 0 :5 :3 5, filter (Aero 0.45 |xm), inject a 50-100 |xL aliquot of th e filtrate.

HPLCVARIABLES

Column: 150 X 3.9 4 [im Nova-Pak C18

Mobile phase: Gradient. A was MeCN. B was MeCN : 150 mM amm onium acetate 10:90.

A:B 0:100 for 10 min, to 30:70 over 10 min, return to initial conditions over 10 min.

Flow rate: 0 .9

Inject ion volume:

50-100

Detector: MS , VG Trio II, probe tip 255, source 180, therm ospray /plasm aspray , m/z 335,

m/z 160


4/11

CHROMATOGRAM

Internal s tandard: penicillin V

OTHER SUBSTANCES


KEYWORDS

cow; SPE; penicillin V is IS

REFERENCE

Boison, J.O.K.; Keng, L.J.-Y; MacNeil, J.D. Analysis of penicillin G in milk by liquid chromatography.

JAOAC Int.,1994,77,565-570

SAMPLE

Matrix: fermentation broth


Adjust pH of fermentation broth to 7, centrifuge at 8000 g for 10

min, add MeCN, centrifuge, add dichloromethane to the supernatant, vortex for 10 s,

shake for 15 min, centrifuge at 8000 g for 15 min. Add 1 mL of the aqueous layer to 100

jiL reagent, heat at 50 for 50 min, cool in an ice bath, inject a 20 fiL aliquot. (Prepare

reagent by dissolving 4.125 g imidazole in 2.5 mL water, add 1 mL HCl, add 500 |xL 110

mM mercury(II) chloride, add 1.5 mL HCl. Recrystallize imidazole twice from

isopropanol.)

HPLC VARIABLES

Guard column:

10 X 4 5 |xm Spherisorb C18

Column:

20 X 4.6 5 |xm Spherisorb C18 S5ODS2

Mobile phase: G radie nt. MeCN :buffer from 16.5:8 3.5 to 31.5 :68.5 over 17 m in (Buffer w as

10 mM NaH

2

PO

4

containing 10 mM EDTA, adjusted to pH 6.5 with 2 M NaOH.)

Flow rate: 2

Inject ion volume: 20

Detector: UV325

CHROMATOGRAM

Retent ion t ime:

14.5


1 jxg/mL

OTHER SUBSTANCES

Extracted:

methicillin, penicillin G, penicillin X

KEYWORDS

derivatization

REFERENCE

Rogers, M.E.; Adlard, M.W.; Saun ders, G.; Holt, G. High-performance liquid chrom atographic determ i-

nation of penicillins following derivatization to mercury-stabilized penicillenic acids. J.Liq.

Chromatogr.,

1983

6, 2019-2031

SAMPLE

Matrix: formulations


Powder 20 tablets, dissolve a portion of the powder in water such

that the concentration of penicillin V potassium is 0.6 mg/mL, mix well, filter. Mix 20 mL

filtrate with 15 mL 0.4 mg/mL sulfadimethoxine in MeCN : wa ter 50:5 0, dilute to 100 mL

with water, mix well, inject a 20 |xL aliquot.

HPLC VARIABLES

Guard column:

40 mm long 30-50 |xm Whatman Co:Pell ODS


5/11

Column:

300 X 3.9 10 |xm ixBondapak C18

Mobile phase:

MeC N.MeO H: 10 mM KH

2

PO

4

2 1 : 4 : 7 5

Flow rate: 1-1.5


Detector:

UV 225

CHROMATOGRAM

Internal s tandard:

sulfadimethoxine

KEYWORDS

tablets; collaborative study

REFERENCE

Mopper, B. Liquid chromatographic determination of penicillin V potassium in tablets: collaborative

study. J.Assoc.Off.Anal.Chem., 1989, 72, 883-884

SAMPLE

Matrix:

formulations

Sample preparat ion: Blend tablets and capsules with water in a high-speed blender for

5 min, filter, dilute with mobile phase, inject a 20 |xL aliquot. Dilute oral suspensions and

injections with mobile phase, inject a 20 |xL aliquot.

HPLCVARIABLES

Guard co lumn: 70 mm long Co:Pell ODS

Column: 300 X 4.6 10 jim Chromegabond C18 (E.S. Indu stries)

Mobile phase:

MeCN : MeO H: 10 mM KH

2

PO

4

19:11:70

Flow rate: 1

Inject ion vo lume: 20

Detector: UV225

CHROMATOGRAM

Retent ion t ime: 8.4


1.02 |xg/mL

OTHER SUBSTANCES

Simul taneous:

amoxicillin, ampicillin, cloxacillin, dicloxacillin, methicillin, nafcillin, oxa-

cillin, penicillin G

KEYWORDS

tablets; capsules; oral suspensions; injections

REFERENCE

Briguglio, G.T.; Lau-Cam, C A . Sep aration and identification of nine pe nicillins by reverse p has e liquid

chromatography. J.Assoc.Off.Anal.Chem., 1984,67, 2 2 8 - 2 3 1

SAMPLE

Matrix:

milk

Sample preparat ion: 10 mL Milk + 2 mL 200 mM tetraethylammonium chloride, stir,

slowly add 38 mL MeCN over 30 s, let stand for 5 min, decant the supernatant through

a plug of glass wool. 40 mL Filtrate + 1 mL water, evaporate under reduced pressure to

1-2 mL, make up to 4 mL with water, filter (0.45 |xm polyvinylidene difluoride). Inject 2

mL in to a n LC system (150 X 4.6 5 |jim Su pelcosil LC-18; M eCN : 10 mM KH

2

PO

4

0:100

for 3 min, to 40:60 over 27 min, to 0:100 over 1 min; 1 mL/min; UV 210 and 295), collect

a 1.5 m L fraction at re ten tion tim e for penicillin V (25 m in), eva por ate to 1 mL , inject a

200 IJLL

aliquot.


6/11

HPLCVARIABLES

Column:

150 X 4.6 5 jxm Supelcosil LC-18-DB

Mobile phase: M eCN : buffer 33:6 7 (Buffer w as 5 mM phosphoric acid an d 5 mM KH

2

PO

4

.)

Flow rate: 1

Inject ion vo lum e:

200

Detector: UV210

CHROMATOGRAM

Limit of quantitation: 2-5

ppb; cow

OTHER SUBSTANCES

Extracted:

amoxicillin, ampicillin, ceftiofur, cephapirin, cloxacillin, penicillin G

REFERENCE

Moats, W.A.; Harik-Khan, R. Liquid chromatographic determination of p-lactam antibiotics in milk: A

multiresidue approach. J.AOAC Int., 1995, 78,4 9 - 5 4

SAMPLE

Matrix:

milk

Sample preparat ion: Add 2 volumes MeCN to milk, stand 5 min, decant aqu eous portion,

suction filter, ex tract w ith an equal volume of dichloro m ethane: hex ane 5 0:50 , centrifuge

aqueous phase at 3000 rpm for 10 min. Dilute 3:1 with 20 mM sodium acetate buffer

and filter (0.2 |xm nylon). Inject 50 |JLL onto column with mobile phase A, run mobile

pha se A for 30 m in an d elu te to w aste. After 30 m in switch to mobile ph ase B a nd elute

through detector.

HPLCVARIABLES

Column: 100 X 8 Ra dial-P ak 10|xm ixBondapak C18

Mobile phase:

A 20 mM sodium a cetate buffer; B G radient. M eCN : M eOH : 20 mM sodium

acetate buffer from 15:10:75 to 30:0:70 over 15 min and hold at 30:0:70

Flo w rate: A 3; B 2


Detector: E, Waters 464 pulsed electrochemical detector, thin layer cell, Ag/AgCl reference

electrode, E l = 1300 mV for 0.166 s, E2 = 1500 mV for 0.166 s, E3 = -200 mV for 0.333 s.

CHROMATOGRAM

Retent ion t ime:

11.8

Limit of detection: 0.2 ppm

OTHER SUBSTANCES

Extracted: am picillin, cloxacillin, dicloxacillin, m ethic illin, nafcillin, oxacillin, pen icillin G

REFERENCE

Kirchmann, E.; Earley, R.L.; Welch, L.E. The electrochemical detection of penicillins in milk.

J.Liq.Chromatogr.,

1994,

17,

1755-1772

SAMPLE

Matrix:

milk


50 g M ilk + 2 drops penicillinase (Difco La bora tories), let stan d 1 h

at 37, add 50 mL MeCN, shake vigorously for 1 min, centrifuge at 9000 g for 10 min,

decant, add 5 g NaCl, swirl to dissolve, add 100 mL dichloromethane, shake for 1 min,

centrifuge at 1000 g for 10 min. Remove top aqueous layer and extract organic layer with

25 mL 10 NaC l by sha kin g and centrifuging as before. Combine aqueou s layers, add 1

mL 0.3 mercu ric chloride in water, let stan d 30 min, add 1 mL 2 M HCl, extract w ith

thr ee 50 mL portions of dichlorom ethane by shak ing each portion for 1 min and eentri-


7/11

fuging at 1000 g for 10 min, filter dichloromethane extracts through 30 g anhydrous

sodium sulfate, evaporate to dryness under reduced pressure at 35, if water remains add

5-10 mL MeOH to flask and com plete evap oration . Dissolve res idu e in 1 mL 10 acetic

acid, add 0.5 mL 0.08 dan syl hy dra zin e in 10 acetic acid, let sta nd 90 m in to over night

in the dark, transfer reaction mixture to a separatory funnel with three 25 mL portions

of dichloromethane, add 5 mL 2 M HCl, shake for 1 min, wash organic layer with 5 mL

5

NaHCO

3

solution, filter thro ugh 10-20 g anhy drou s sodium sulfate. E xtrac t acid aque-

ous layer again with 25 mL dichloromethane. Combine dichloromethane layers and evap-

orate to dryness at 35 under reduced pressure. Dissolve residue in 2 mL IS solution,

inject a 20

JJLL

aliquot. (Pr epa re IS so lution by dissolving 10 JXLbenzaldeh yde in 100 mL

dichloromethan e, evapo rate 1 mL to dryn ess und er reduced pres sur e, dissolve residu e in

1 mL 10 acetic acid, add 0.5 mL 0.08 dan syl hydrazine in 10 acetic acid, let sta nd

90 min to overnight in the dark, transfer reaction mixture to a separatory funnel with

three 25 mL portions of dichloromethane, add 5 mL 2 M HCl, shake for 1 min, wash

organic layer with 5 mL 5 NaH CO

3

solution, filter through 10-20 g anhydrous sodium

sulfate. Extract acid aqueous layer again with 25 mL dichloromethane. Combine dichlo-

romethane layers and evaporate to dryness at 35 under reduced pressure. Dissolve res-

idue in 100 mL MeCN then dilute an aliquot 1:4 with MeCN.)

HPLC VARIABLES

Column: 250 X 4 10 jxin Lichrosorb RP-18

Mobile phase:

MeCN: water 58:42

Flow rate: 1

Inject ion volume:

20

Detector: F ex 254 em 500 (filter)

CHROMATOGRAM

Retent ion t ime:

6.73

Internal s tandard: benzaldehyde (derivatized) (12.18)

Limit of detection: 5

ng/g

OTHER SUBSTANCES

Extracted: cloxacillin, dicloxacillin, methicillin, nafcillin, penicillin G, phenethicillin

Interfering:

oxacillin

KEYWORDS

derivatization

REFERENCE

Munns, R.K.; Shimoda, W.; Roybal, J.E.; Vieira, C. Multiresidue method for determination of eight

ne utr al (3-lactam penicillins in milk by fluorescence-liquid chroma tography. J.Assoc.Off.Anal.Chem.,

1985 68, 9 6 8 - 9 7 1

SAMPLE

Matrix: milk


Condition a Sep-Pak C18 SPE cartridge with 20 mL MeOH

5

20 mL

water, and 2 mL 2 NaC l. Pa ss throu gh 30 g filtered (glass-wool plug) m ilk at 2 mL /min,

wash with 5 mL water, wash with 10 mL M eOH: w ater: 20 NaCl 10 :80:10 containing

20 mM 18-crown-6, elu te w ith 10 mL 15 (v/v) M eOH , inject a 100 fxL aliquo t.

HPLCVARIABLES

Guard column:

50 X 2.1 Permaphase ETH (Du Pont)

Column: 150 X 4.3 LiC hrosorb R P-18

Mobile phase: M eO H:w ater:0.2 M pH 4.0 phosp hate buffer 25 :65 :10 con taining 11 mM

sodium 1-heptanesulfonate

Column temper ature: 45

Flow rate: 1


8/11

Injection volume: 100

D etec tor: UV 210

C H R O M A T O G R A M

Retention time: 18.5

Lim it of dete ction : 30 ng/g

OTHER SUBSTANCES

E xt ra ct ed : ampicillin, penicillin G

KEYWORDS

cow; SPE

REFERENCE

Terada, H.; Sakabe, Y. Studies on residual antibacterials in foods. IV. Simultaneous determination of

penicillin G, penicillin V and ampicillin in milk by high-performance liquid chromatography.

J.Chromatogr., 1985 348, 379-387

SAMPLE

Matrix: solutions

Sa mple p re p ara ti o n : Separate buffer containing drug from human serum albumin by cen-

trifuging at 37 at 700 g for 3 min using a Micropartition System MPS-I (Amicon) unit,

inject a 10-20 fxL aliquot of the ultrafiltrate.

HPLC VARIABLES

Guard column: C18/Corasil (Waters)

Column: 300 X 3.9 ixBondapak C18

Mobile p ha se : MeCN: 10 mM ammonium acetate 25:75

Flow rate: 1.5

Injection volume: 10-20

D etec tor: UV 220

REFERENCE

Terasak i, T.; Nouda, H.; Tsuji, A. Selective analysis of mutu al displaceme nt effects at th e prim ary bind-

ing sites of phenoxymethylpenicillin and cephalothin bindings to human serum albumin.

J.Pharmacobiodyn., 1992 15, 9 1 - 9 7

SAMPLE

Matrix: solutions

Sa mple p rep ara ti on : Prepare an aqueous solution, inject a 200 |xL aliquot.

HPLCVARIABLES

Guard column: present but not specified

Colu mn: 150 X 4.6 4 ^m Micropak SPC-18 C18

M obile phase : Gradient. MeCN: 10 mM orthophosphoric acid from 15:85 to 60:40 over 20

min

Flow rate: 1

Injection volume: 200

Detec tor: UV 220


Retention time: 15

OTHER SUBSTANCES

Sim ultaneous: carbenicillin, cloxacillin, dicloxacillin, methicillin, nafcillin, penicillin G


9/11

REFERENCE

Moats, W.A. Effect of the silica support of bonded reversed-phase columns on chromatography of some

antibiotic compounds.

J.Chromatogr.,

1986

366,

6 9 - 7 8

SAMPLE

Matrix:

tissue

Sample preparat ion: Homogenize (Ultra-Turrax) 25 g tissu e with 25 mL MeCN for 1 min,

add 5 mL 500 mM pH 2.2 phosphate buffer while the homogenizer is still running, add

65 mL MeCN, homogenize for 1 min, centrifuge at 4000 g for 10 min. Remove the super-

natant and add it to 7 g NaCl and 50 mL dichloromethane, shake for 2 min, allow to

stand for 30 min. Remove the upper organic layer and add it to 5 g anhydrous sodium

sulfate, shake for 30 s, filter through a cotton-wool plug, evaporate to about 4 mL under

reduced pressure at 30, add 3 mL dichloromethane, evaporate to about 4 mL, add 3 mL

light petroleum, evaporate to about 0.5 mL, Suspend this residue with sonication in three

3 mL portions of light petroleum and place these fractions in a separate tube, rinse the

original tube with 2 mL pH 7 phosphate buffer. Add the phosphate buffer rinse to the

light petroleum extracts, vortex for 30 s, centrifuge, remove the aqueous layer. Extract

the light petroleum layer with 2 mL pH 7 pho sph ate buffer and with two 1.5 mL portions

of pH 7 phosphate buffer, combine all the aqueous phases, centrifuge, inject a 200 (JLL

aliquot onto column A and elute to was te with mobile phas e B, after 15 min e lute to was te

with m obile phase C at 2 m L/min, after 10 min elute th e con tents of column A onto column

B with mobile phase D, after 2 min rem ove column A from th e circuit, elute column B

w ith mobile phas e D, m onitor t he effluent from column B . (Wash column A w ith m obile

pha se A at 2 mL /min for 7 min, with mobile phase A at 1 mL/m in for 5 min, w ith mobile

pha se B at 2 mL/min for 8 min, an d w ith mobile ph ase B at 1 mL /min for 6 min.)

HPLCVARIABLES

Column: A 4 X 4 5 | x m LiChrospher 100 RP-18e; B 250 X 4 5 |xm LiChrospher 100 RP-18e

Mobile phase: A M eCN: water 50:50; B 20 mM pH 7 phosphate buffer; C MeCN : 20 mM

pH 3 pho sph ate buffer 10:90; D M eCN : 200 mM pH 3.0 phos pha te buffer 35:6 5 contain-

ing 2 mM disodium EDTA

Column temperature: 35

Flow rate:

1 (except wh ere ind icated)

Inject ion vo lume: 200

Detector:

E, Merck Model L3500, glassy carbon working electrode +0 .65 V, stainless-steel

auxiliary electrode, Ag/AgCl reference electrode following post-column reaction. The col-

umn effluent flowed through a 10 m X 0.3 mm ID woven PTFE coil illuminated by a UV

254 low-pressure mercury lamp to the detector.

CHROMATOGRAM

R eten t ion t ime: 6.1

Limit of detection: 1.4

ng

OTHER SUBSTANCES

Extracted:

cloxacillin, dicloxacillin, oxacillin, penicillin G

KEYWORDS

post-column reaction; cow; muscle; column-switching

REFERENCE

Lihl, S.; Rehorek, A.; Petz, M. High-performance liquid chrom atographic determ ination of penicillins by

means of automated solid-phase extraction and photochemical degradation with electrochemical de-

tection.J.Chromatogr.A, 1996, 729, 2 2 9 - 2 3 5

SAMPLE

Matrix:

tissue


10/11


Blend 15 g tissu e with 45 mL (60 mL for liver and kidney) water in

a 300 or 500 mL blender jar at half power (or less to control foaming) for 2 min. Add a

20 mL aliquot of homogenate to 40 mL MeCN, mix, after 5 min decant supernatant

throu gh a plug of glass wool, collect 30 mL. Sh ake vigorously 30 mL n itra te, 10 mL 200

mM phosphoric acid, and 20 mL dichloromethane, remove organic layer and extract aque-

ous layer with 10 mL dichlorom ethane (and 10 mL MeCN for liver and kidneys). Combine

dichloromethane layers, add 15 mL MeCN, add 40 mL hexane, wash the mixture twice

with 4 mL portions of water, ex tract th e organic layer four time s with 1 mL 10 mM pH

7 buffer. Combine extracts and add 0.1-0.2 mL tert-butanol, place in a rotary evaporator

without heating at first. When the flask becomes cold warm to 50, concentrate to less

th an 1 mL , adjust to a final volum e of 1 mL , filter (Gelm an Acrodisc LCPVDF), inject a

200

|JLL

aliquot.

HPLCVARIABLES

Guard column:

Polymer Labs guard cartridge

Column: 150 X 4.6 5 jxm 100 A PLRP-S polystyrene-divinylbenzene (Polymer Labs)

Mobile phase:

M eCN : buffer 18:82 , after ru n w as over flush at 35 :65 for 5 min th en re-

equilibrate with 18:82 for 9 min. (Buffer was 10 mM pH 7 phosphate buffer prepared

from 1.36 KH

2

PO

4

and 2.84 g Na

2

HPO

4

in 3 L water.)

Flow rate: 1


Detector: UV

210

CHROMATOGRAM

Retent ion t ime: 9-11


10 ng/g

KEYWORDS

cow; pig

REFERENCE

Moats, W.A. High-performance liquid chromatographic determination of penicillin G, penicillin V and

cloxacillin in beef and pork tissues.J.Chromatogr., 1992 593, 1 5 - 2 0

SAMPLE

Matrix:

tissue


Condition a 6 mL 500 mg Bond Elu t C18 SPE cartridg e with 20 mL

MeOH, 20 mL water, an d 10 mL 2 NaC l, do not allow to go dry. 5 g Tissue + 20 mL

water, homogenize (Polytron, 20 mm probe), rinse probe with water so that total volume

is 35 mL, shake mechanically for 5 min, add 5 mL 170 mM sulfuric acid, add 5 mL 5

sodium tungstate, vortex for 20 s, centrifuge at 2200 g for 10 min, remove the superna-

tant, add 15 mL water to the residue, shake for 5 min, centrifuge at 2200 g for 10 min.

Combine the sup ern atan ts and filter (Whatman GF/B) them , add 10 mL 20 NaCl to the

filtrate, mix thoroughly, add to the SP E ca rtridge at 3 mL/min, was h with 10 mL 2

NaCl, wash with 10 mL water, draw air through the cartridge for 5 min, immediately

elute with 1 mL MeCN:200 mM pH 6.5 sodium phosphate buffer:water 60:5:35. Add 1

mL reag ent to the eluate , vortex, hea t a t 65 for 30 min, cool rapidly to room te m pe ratu re,

vortex, filter (Aero 0.45 |xm), inject a 80-100 |xL aliquot of the filtrate. (Prepare reagent

by dissolving 34.45 g1 2 4-triazolein 150 mL w ater, add 25 mL 10 mM mercu ric chloride

solution, mix, adjust pH to 9.0 0.5 with 5 M NaOH, make up to 250 mL with water.)

HPLCVARIABLES

Column:

150 X 3.9 4 |xm Nova-Pak C18

Mobi le phase: M eCN : buffer 25 :75 (Buffer w as 4.969 g Na

2

HPO

4

,8.969 g NaH

2

PO

4

-H

2

O,

and 2.482 g anhydrous sodium thiosulfate in 1 L water.)

Flow ra te:

0.8


11/11

Injection volume: 80-100

Detecto r: UV 325


Retention time: 7.6

In te rn al sta nd ar d: penicillin V

OTHER SUBSTANCES

E xt ra cted : penicillin G

Simultaneous: ampicillin, chloramphenicol

KEYWORDS

muscle; liver; kidney; derivatization; cow; SPE; penicillin V is IS

REFERENCE

Boison, J.O.; Salisbury, C.D .C; Cha n, W.; MacNeil, J.D. De term ination of penicillin G residues in edible

animal tissues by liquid chromatography. J.Assoc.Off.Anal.Chem., 1991 74, 4 9 7 - 5 0 1

ANNOTATED BIBLIOGRAPHY

Blanchflower, W.J.; Hewitt, S.A.; Kennedy, G. Confirmatory assay for the simultaneous determination

of five penicillins in muscle, kidney and milk using liquid chromatography-electrospray mass spec-

trometry. Analyst,

1994

119, 2595-2601 [LC-MS; meat; LOD 5-100 ng/g; extracted cloxacillin, di-

cloxacillin, oxacillin, penicillin G; nafcillin (IS)]

Orford, CD.; Perry, D.; Adlard, M.W. The determination of naturally produced penicillins and their

biosynthetic precursors using pre-column derivatisation with dansylaziridine. J.Liq.Chromatogr.,

1991 14, 2665-2684 [also penicillin G, penicillin K, penicillin X; LOD 1000 ng/mL; fluorescence

detection]

Martin, J.; Mendez, R.; Negro, A. Effect of temperature on HPLC separations of penicillins.

J.Liq.Chromatogr., 1988 11, 1707-1716 [also amoxicillin, ampicillin, cloxacillin, piperacillin, peni-

cillin G; column temp 15-55]

Mopper, B. Liquid chromatographic determination of penicillin V potassium in tablets and powders for

oral solution.J.Assoc.OffAnal.Chem. 1987 70 3 9 - 4 1

Moller, J.; Hiddessen, R.;

Niehoff

J.; Schiigerl, K. On-line high-performance liquid chroma tography for

monitoring fermentation processes for penicillin production. Anal.Chim.Acta, 1986 190, 195-203

[simultaneous impurities]

Moats, W.A. Determination of penicillin G, penicillin V, and cloxacillin in milk by reversed-phase high-

performance liquid chromatography. J.Agric.Food Chem., 1983 31, 880-883 [gradient; LOD 5 ppb;

extracted cloxacillin, penicillin G]

LeBeIIe, M.J.; Lauriault, G.; Wilson, W.L. High performance liquid chromatographic analysis of peni-

cillin V solid dosage forms. J.Liq.Chromatogr.,

1980

3, 1573-1578

Documents

Penicillin V