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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
Sample preparat ion:
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)
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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
Sample preparat ion:
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
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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
Extracted: penicillin G
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
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CHROMATOGRAM
Internal s tandard: penicillin V
OTHER SUBSTANCES
Extracted: penicillin G
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
Sample preparat ion:
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
Limit of detect ion:
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
Sample preparat ion:
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
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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
Inject ion volume: 20
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
Limit of detect ion:
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.
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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
Inject ion volume: 50
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
Sample preparat ion:
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-
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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
Sample preparat ion:
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
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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
C H R O M A T O G R A M
Retention time: 15
OTHER SUBSTANCES
Sim ultaneous: carbenicillin, cloxacillin, dicloxacillin, methicillin, nafcillin, penicillin G
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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
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Sample preparat ion:
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
Inject ion volume: 200
Detector: UV
210
CHROMATOGRAM
Retent ion t ime: 9-11
Limit of detect ion:
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
Sample preparat ion:
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
7/25/2019 Penicillin V
11/11
Injection volume: 80-100
Detecto r: UV 325
C H R O M A T O G R A M
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