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Accepted Manuscript
Title: A simple LC-MS/MS method using HILICchromatography for the determination of fosfomycin inplasma and urine: application to a pilot pharmacokinetic studyin humans
Author: Suzanne L. Parker Jeffrey Lipman Jason A. RobertsSteven C. Wallis
PII: S0731-7085(14)00578-0DOI: http://dx.doi.org/doi:10.1016/j.jpba.2014.11.042Reference: PBA 9829
To appear in: Journal of Pharmaceutical and Biomedical Analysis
Received date: 17-10-2014Revised date: 21-11-2014Accepted date: 23-11-2014
Please cite this article as: S.L. Parker, J. Lipman, J.A. Roberts, S.C. Wallis, Asimple LC-MS/MS method using HILIC chromatography for the determinationof fosfomycin in plasma and urine: application to a pilot pharmacokineticstudy in humans., Journal of Pharmaceutical and Biomedical Analysis (2014),http://dx.doi.org/10.1016/j.jpba.2014.11.042
This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.
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ICU patients
plasma and urine
pharmacokinetic profile
HILIC and LC-MS/MS bioanalysis
Fosfomycin
*Graphical Abstract
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1
A simple LC-MS/MS method using HILIC chromatography for the 2
determination of fosfomycin in plasma and urine: application to a pilot 3
pharmacokinetic study in humans. 4
Suzanne L. Parker1,a, Jeffrey Lipmana,b, Jason A. Robertsa,b , Steven C. Wallisa 5
a Burns, Trauma and Critical Care Research Centre, The University of Queensland, 6
Brisbane, Australia 7
b Royal Brisbane and Women’s Hospital, Brisbane, Australia 8
1Author for correspondence: Tel: +6173346 5104 9
E-mail: [email protected] 10
Present address: School of Medicine, The University of Queensland, Herston, 11
Queensland, AUSTRALIA 12
13
14
Keywords: 15
Fosfomycin; Antibiotic; LC-MS/MS; Pharmacokinetic; HILIC 16
17
18
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Abstract 20
A high performance liquid chromatography - tandem mass spectrometry (LC-MS/MS) 21
method, using hydrophilic interaction liquid chromatography (HILIC) 22
chromatography for the analysis of fosfomycin in human plasma and urine, has been 23
developed and validated. The plasma method uses a simple protein precipitation 24
using a low volume sample (10 μL) and is suitable for the concentration range of 1 to 25
2000 μg/mL. The urine method involves a simple dilution of 10 μL of sample and is 26
suitable for a concentration range of 0.1 to 10 mg/mL. The plasma and urine results, 27
reported respectively, are for recovery (68, 72%), inter-assay precision (≤9.1%, 28
≤8.1%) and accuracy (range -7.2 to 3.3%, -1.9 to 1.6%), LLOQ precision (4.7%, 3.1%) 29
and accuracy (1.7% and 1.2%), and includes investigations into the linearity, stability 30
and matrix effects. The method was used in a pilot pharmacokinetic study of a 31
critically ill patient receiving IV fosfomycin, which measured a maximum and 32
minimum plasma concentration of 222 μg/mL and 172 μg/mL, respectively, after the 33
initial dose, and a maximum and minimum plasma concentration of 868 μg/mL and 34
591 μg/mL, respectively, after the fifth dose. The urine concentration was 2.03 35
mg/mL after the initial dose and 0.29 mg/mL after the fifth dose. 36
37
HIGHLIGHTS 38
A simple and robust LC-MS/MS method for the quantification of fosfomycin 39
in human plasma and urine has been developed. 40
The method uses HILIC chromatography that supports a simple treatment of 41
fosfomycin from biological fluids. 42
The developed LC–MS/MS method has been validated according to published 43
US FDA guidelines and shows excellent performance. 44
Results of a critically ill patient from a pilot pharmacokinetic study with 45
receiving IV fosfomycin are included. 46
This is the first method published that is suitable for the quantification of 47
fosfomycin in both plasma and urine. 48
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50
1. Introduction 51
Fosfomycin (FOM) is a broad-spectrum antibiotic that is generating substantial 52
interest as an intravenous or enteral therapy for the treatment of multi-drug 53
resistant (MDR) infections [1, 2]. Antibiotic resistance is a significant and immediate 54
global health concern and an increasing prevalence of MDR bacteria is steadily 55
decreasing the number of usable antibiotics available [3]. In this context, fosfomycin 56
represents an important treatment option. 57
FOM was first discovered in Spain in 1969 and is used as treatment therapy for 58
uncomplicated urinary tract infections [4] and often combined with beta-lactams or 59
aminoglycosides for a synergistic effect against Pseudomonas aeruginosa [5, 6]. FOM 60
is structurally unrelated to other antibiotics: it is a small (138 Da), highly hydrophilic 61
phosphonic acid and, with negligible protein binding [7], exhibits excellent 62
penetration into tissue [8]. FOM has a unique mechanism of action by way of its 63
ability to inhibit the synthesis of peptidoglycan found in the inner cell wall of Gram-64
negative and Gram-positive bacteria [9]. These characteristics support the 65
effectiveness of FOM for the treatment of MDR pathogens and it has been used 66
extensively as a last-line antibiotic for treatment of critically ill patients [2]. 67
Investigating the pharmacokinetic (PK)/pharmacodynamic (PD) characteristics of 68
FOM may enable development of robust dosing strategies that maximize the PD 69
response to treatment while minimizing the potential future development of 70
antibiotic resistance. A reliable method of quantification of FOM in plasma is needed 71
to define the pharmacokinetic profile of the drug and urine data can provide 72
valuable information on elimination rates. The information obtained can be used to 73
characterize the PK/PD relationship. 74
There are several analytical techniques available for the determination of FOM in 75
biological fluids, using gas chromatography [10-14], liquid chromatography (LC) - 76
spectrophotometric detection [15], LC - photometric detection [16], capillary zone 77
electrophoresis [17, 18], and, more recently, with derivatization and LC - 78
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atmospheric pressure chemical ionization mass spectrometry [19] and LC – tandem 79
mass spectrometry (MS/MS) [20-22]. 80
A method suitable for use in a pharmacokinetic study with patients receiving 81
multiple intravenous doses, up to around 12-24 g/day (as are now being used 82
clinically [2]) would require a lower limit of quantification (LLOQ) of around 1 μg/mL 83
for plasma and 0.1 mg/mL for urine. However, patients with renal insufficiency or 84
with altered pharmacokinetics – as is commonly seen in the critically ill – receiving 85
multiple doses of antibiotics can exhibit very high concentrations in their plasma. 86
This has, therefore, led to an unusually extended concentration range (from 1 to 87
2000 μg/mL for plasma) being described here, which is supported by the data from 88
the pilot study. While there are many methods currently available for the analysis of 89
FOM in biological fluids, and the method by Li offers a rapid and sensitive alternative 90
for plasma [20], we are unaware of any methods suitable for the analysis of FOM in 91
both plasma and urine that offer the concentration range to meet these clinical 92
specifications at this time. 93
This paper describes an analytical technique using hydrophilic interaction 94
chromatography (HILIC) – tandem mass spectrometry that offers a simple and 95
reliable determination of FOM in plasma and urine, with a quick and reproducible 96
sample preparation. 97
2. Experimental 98
2.1. Chemicals and materials 99
Fosfomycin (FOM), ethylphosphonic acid (EPA, internal standard) and acetonitrile 100
(HPLC gradient-grade solvent) were purchased from Sigma-Aldrich and ammonium 101
acetate was obtained from Ajax Univar. Ultra-pure water was obtained using a 102
Permutit system. Drug-free human plasma was obtained from the Australian Red 103
Cross Blood Service and drug-free urine was obtained from healthy volunteers. 104
2.2. Instrumentation and conditions 105
The LC-MS/MS used is a Shimadzu Nexera UHPLC equipped with a triple quadrupole 106
8030+ Shimadzu mass spectrometer (MS) detector. An electro-spray ionization (ESI) 107
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source interface operating in negative-ion mode was used for the multiple reaction 108
monitoring (MRM) LC-MS/MS analysis. MS conditions for FOM and the internal 109
standard (IS; EPA) are reported in Table 1. The interface settings consisted of the 110
nebulizing gas flow of 3 L/min, a de-solvation line temperature of 250°C, heat block 111
temperature of 400°C and a drying gas flow of 15 L/min. 112
The compounds were separated on a Merck SeQuant zic-HILIC, 2.1 x 50 mm, 5.0 μm 113
analytical column (operated at 24° C) protected by a 20 mm SeQuant zic-HILIC 114
guard cartridge using an isocratic mobile phase containing acetonitrile with 2 mM 115
ammonium acetate, pH 4.8 (85/15 v/v) at a flow rate of 0.3 mL/min. The injection 116
volumes used were 0.1 μL for the plasma assay and 0.5 μL for the urine assay. The 117
retention time for both FOM and EPA was 2.5 min. 118
2.3. Stock and standard solution preparation 119
2.3.1. Standards for plasma analysis 120
Aqueous stock solutions for plasma standard preparation (at 1, 2 and 10 mg/mL) 121
were stored at -80°C. On the day of assay these were diluted with drug free plasma 122
to yield calibration standards from 1 to 2000 µg/mL that was processed alongside 123
the clinical samples. 124
2.3.2. Standards for urine analysis 125
Aqueous stock solutions for urine standards of FOM (2, 5, 10, and 50 mg/mL) were 126
stored at -20°C. On the day of assay these were diluted with drug free urine to 127
prepare calibration standards from 0.1 to 10 mg/mL that were processed alongside 128
the clinical samples. 129
2.3.3. Internal standard solution 130
Ethylphosphonic acid (EPA) in acetonitrile was used as internal standard for the 131
plasma assay (10 µg/mL), and an aqueous EPA solution was used as internal standard 132
for the urine assay (1 mg/mL). Solutions were stored at -20°C. 133
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2.3.4. Quality Controls 134
Quality controls were prepared by spiking drug free plasma with FOM to 135
concentrations of 3, 800 and 1600 µg/mL, and stored at -80°C until assay. On the day 136
of assay an additional QC at 80 µg/mL was prepared by diluting with blank plasma 137
the QC at 800 µg/mL. The four sets of QCs were assayed alongside clinical samples. 138
Quality controls for urine analysis were prepared at FOM concentrations of 0.3, 2 139
and 8 mg/mL. The urine QCS were stored at -20°C until assayed alongside clinical 140
samples. 141
2.4. Analytical procedure 142
2.4.1. Clinical plasma sample preparation 143
Clinical samples were prepared by combining 10 μL of clinical sample, 10 μL of 144
water, and 90 μL of drug-free blank plasma. Internal standard (300 μL, 10 μg/mL of 145
EPA in acetonitrile) was then added and the sample vortexed and then centrifuged 146
(for 5 min at 14,000 rpm) to remove precipitated proteins. A volume of 0.1 μL was 147
injected onto the LC-MS/MS system. 148
2.4.2. Clinical urine sample preparation 149
All urine samples were filtered using a 0.22 µm filter prior to use. Clinical samples 150
were prepared by combining 10 μL of sample with 10 μL water, followed by internal 151
standard (20 μL, 1 mg/mL of EPA). The sample was then diluted with 200 μL of 152
mobile phase and 0.5 μL was injected into the LC-MS/MS system. 153
2.4.3. Data analysis 154
For both plasma and urine the concentration of each clinical sample was obtained 155
using the data from the calibration curve prepared (in either plasma or urine) within 156
the batch using a quadratic regression with peak-area ratio (drug/internal standard 157
area responses) against concentration (x), with a 1/x2 weighting as the mathematical 158
basis of the quantification. 159
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2.5. Validation 160
The validation was performed in accordance with the guidelines provided by the US 161
Food and Drug Administration (USFDA) and met the criteria required to demonstrate 162
the method is suitable for intended purpose [23]. The validation for both plasma and 163
urine was assessed for matrix effects, lower limit of quantification (LLOQ), linearity, 164
inter-day precision and accuracy, freeze-thaw stability of quality control samples and 165
the stability of standard solutions. 166
2.5.1. Limit of quantification 167
The lower limits of quantification for FOM were evaluated by analysis of replicate 168
standards, for both plasma and for urine samples. 169
2.5.2. Linearity 170
To investigate linearity, calibration curves were prepared using the corresponding 171
concentration ranges suitable for each matrix. 172
2.5.3. Inter-day precision and accuracy 173
Precision and accuracy for FOM throughout the calibration range of both plasma and 174
urine was evaluated by the analysis of QC samples at four different concentrations 175
for plasma and three different concentrations for urine with the QC concentrations 176
determined against freshly prepared standard curves. In addition to precision and 177
accuracy data obtained from QC samples, an incurred sample reanalysis was 178
performed. 179
Acceptance criteria were applied according to the US Food and Drug Administration 180
guidelines [23]; with acceptance criteria on an incurred sample reanalysis applied 181
according to the European Medicines Agency guidelines [24]. 182
2.5.4. Matrix effects 183
Plasma matrix effects were evaluated to identify any suppression or enhancement of 184
signal from an interfering substance around the retention time of FOM and EPA by 185
using the matrix factor test. Five blank plasma samples were assayed at spiked low 186
and high concentration levels and with internal standard, and the area results 187
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compared to those produced following the same extraction procedure using water 188
instead of plasma. The precision of the matrix factor (normalized against the internal 189
standard) was used to determine if any concentration level demonstrated a trend of 190
variation from the expected result. 191
Five urine blanks were assayed at a spiked low and high concentration level and the 192
precision and accuracy calculated, with respect to the nominal concentrations, to 193
determine if any concentration level demonstrated a trend of variation from the 194
expected result. 195
2.5.5. Recovery 196
The recovery of FOM and EPA was evaluated by comparing the peak area for plasma 197
or urine samples spiked prior to protein precipitation (for plasma) or dilution (for 198
urine) with samples spiked after protein precipitation or dilution. Care was taken to 199
ensure the injection matrix was identical in comparable samples. 200
2.5.6. Stability 201
Stability of FOM in plasma and urine was assessed across three freeze-thaw cycles 202
(from -80°C to ambient temperature) using three replicates of the QC samples at 203
low, medium and high concentrations. Stability of stock solutions was assessed 204
comparing aqueous solutions stored at both -20°C and -80°C to freshly prepared 205
solutions. 206
2.6. Pharmacokinetic application 207
The method was developed for the analysis of plasma and urine samples from a 208
pharmacokinetic study with critically ill patients receiving an intravenous dose of 6 g 209
of FOM every 6 hours with expected peak plasma concentrations of around 200 210
µg/mL, an expected plasma half-life of 2 h, and urinary concentrations of around 5 211
mg/mL [25]. 212
One critically ill patient was administered an intravenous dose of 6 g fosfomycin 213
disodium. Blood samples (3 mL) were taken prior to dosing (0 h) and 0.5, 0.75, 1, 1.5, 214
2, 4, and 6 h post administration using heparinized vacuum tubes (Greiner Bio-One, 215
Vacuette® LiHep) on the first day of FOM administration and after receiving the fifth 216
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FOM dose. Blood samples were centrifuged at 3000 rpm for 10 min to obtain plasma 217
samples. Plasma samples were transferred into 2 mL polypropylene tubes, capped 218
and stored at -80°C until analysis. 219
Similarly, a urine sample was collected 6 h post administration of the FOM dose. The 220
urine was transferred into a urine specimen vial, capped and stored at -80°C until 221
analysis. 222
This procedure was conducted in accordance with the principles laid down by the 223
ICH guidelines for Good Clinical Practice and approved by the University of 224
Queensland Medical Research Review Committee (clearance # 2012000870) and the 225
Epistimoniko Symvouleio (Scientific Committee) of Attikon University Hospital 226
(approval MEΘ-84/13-3-12). 227
3. Results and discussion 228
3.1. Chromatography 229
This method has been established using HILIC technology which offers excellent 230
selectivity for polar hydrophilic compounds like FOM, and the use of a high organic-231
solvent content in the mobile phase leads to a rapid evaporation of the solvent 232
during electrospray ionization [26], endowing the method with a simple 233
compatibility with mass spectrometry. Additionally, the use of HILIC technology in 234
this method obviates the requirement for further modification of the sample, after a 235
solvent-based protein precipitation, to closely correspond with the organic content 236
of the mobile phase for improved peak shape. This simplicity of extraction leads to 237
low detection levels when using low sample volumes. 238
Retention of the analyte by the stationary phase is caused by hydrophilic partitioning 239
within an aqueous-enriched liquid layer and/or with the positive or negative charges 240
on the HILIC stationary phase [27]; the balance of the partition is provided by the 241
aqueous content and pH of the mobile phase. Therefore, the aqueous layer is critical 242
to the efficiency of HILIC separation. Published HILIC methodology often 243
recommends mobile phases consisting of high ionic strength (from 5 – 20 mM, with 244
upper limits of 200-300 mM) [28] but this presented difficulties in development. The 245
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use of mobile phases with high ionic strength of buffers, low and variable aqueous 246
content, combined with the high pressure applied in an HPLC system led to blocking 247
of the column and the capillary in the ionization source on the mass spectrometer. 248
This isocratic method uses a low ionic strength of 2 mM ammonium acetate buffer in 249
85% acetonitrile which has been reliable and provided consistent results with 250
minimal loss to chromatographic shape and reproducibility. Regenerating the 251
column after every 300 – 400 injections of samples, particularly the urine samples, 252
and keeping buffer concentration to as low an ionic strength as possible, was 253
advantageous to long term use. Using a guard column extended the column life but 254
as the separation is highly dependent on the salt concentration and its impact on the 255
stationary phases aqueous-rich layer, the regeneration of the column was critical to 256
maintaining the quality of chromatography. 257
Another aspect of the method development that was found to affect both the 258
chromatographic retention and peak shape was the injection volume and the 259
composition of the sample being injected. Despite the low injection volumes being 260
used, 0.1 μL for plasma and 0.5 μL for urine, we conclude that the changes in the 261
quality of the chromatography observed were due to the sensitivity of the 262
interaction of FOM with the stationary phase from changes in ionic strength and pH 263
of the buffer [26, 27]. For the development of a bio-analytical assay using HILIC 264
chromatography, consideration of the organic to aqueous ratio, concentration of 265
salts, and finally, the presence of acids or base, is required. A dilution with plasma 266
was used in the plasma method as the ratio of acetonitrile to aqueous concentration 267
had an impact on the quality of the chromatographic peak shape and retention; the 268
concentrations found in clinical samples from the pilot study allowed this dilution. 269
The urine method included a dilution of sample that improved the chromatographic 270
separation by either reducing the presence of endogenous salts in the sample or 271
controlling the pH. 272
Buszewski [26] and Alpert describe the mechanism of HILIC separation as being 273
based on an interplay of a partitioning equilibrium in the aqueous layer (based on 274
the hydrophilicity of the analyte), weak electrostatic mechanisms, and dipole-dipole 275
interactions (including hydrogen-bonding) [29] the impact of each parameter on the 276
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selectivity and reproducibility of chromatography requires a more sophisticated 277
management than in general reversed-phase chromatography, but which once 278
overcome can lead to a highly stable and robust method. 279
3.2. Validation 280
The LLOQ was determined as 1 μg/mL for plasma and 0.1 mg/mL urine with 281
precision calculated as 4.7 and 3.1%, respectively, and accuracy calculated as 1.7 and 282
1.2%, respectively (Table 2). The signal to noise ratio of the lowest standard in the 283
calibration curve was 23.2 for plasma and 178 for urine and this data, combined with 284
the excellent precision and accuracy obtained at 1 μg/mL for plasma and 0.1 mg/mL 285
for urine, suggests substantial scope for achieving a lower LLOQ for both matrices 286
(see Figure 2 and 3 for a representative chromatogram of the LLOQ standard 287
extracted from plasma and urine, respectively). The lower limit of detection (LLOD) is 288
defined as being reliably distinguished from the background noise and calculated as 289
≥ three-times the noise of the blank plasma sample. From the validation the LLOD 290
was determined as being approximately 0.01 μg/mL for plasma and <0.01 mg/mL for 291
urine. 292
A regression model with a weighted (1/x2) quadratic curve provided the lowest 293
distribution of error across the substantial concentration range (1 to 2000 μg/mL for 294
plasma and 0.1 to 10 mg/mL for urine). The results of the linearity study are 295
described in Table 3. 296
Precision and accuracy of the QC samples are shown in Table 4 for both plasma and 297
urine. All results were within the acceptance criteria of ± 15% of the nominal 298
concentration, indeed the results of all plasma QCs samples were within 9.1% and 299
urine within 4.2%. An incurred sample reanalysis was performed on a subset of 300
clinical samples and the results meet the current guidelines [23, 24] with >67% of 301
repeated results being within 30% of the mean. Indeed, 100% of the repeated results 302
were within 11%. 303
No signal suppression/enhancement was evident for either FOM or the internal 304
standard from the matrix study performed. The matrix effect evaluation is reported 305
in Table 5. 306
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Despite using a very simple protein-precipitation for the extraction of FOM from 307
plasma the recovery was somewhat low at 68%. However, this extraction recovery is 308
not atypical for a drug with a highly hydrophilic nature due to preferential aqueous 309
partitioning. As was seen from the LLOQ testing, the variability was reliable 310
(precision 6.1%) and sensitivity (LLOD ca. 0.01 μg/mL) easily achievable. The recovery 311
of the internal standard EPA was good at 72% when tested at the undiluted 312
concentration of 10 μg/mL. The urine preparation was a simple dilution with internal 313
standard and as such provided recoveries of 98% when tested at 0.4 mg/mL. The 314
recovery results are reported in Table 5. 315
Stock solution stability for FOM was tested for aqueous solutions stored for over 16 316
months at -80°C and for over 11 months at -20°C and it was found to be stable. FOM 317
was also found to be stable in plasma and urine across three freeze-thaw cycles 318
when stored at -80°C and thawed at ambient temperature in water (see Table 5). 319
Overall, the validation of this method was highly successful for both plasma and 320
urine with the method showing an excellent degree of reproducibility and accuracy, 321
and is suitable for use in the analysis of patient samples in a pharmacokinetic study. 322
This technique offers a simple and robust method for the analysis of FOM in both 323
plasma and urine in patient samples. Other quantitative methods have been 324
described for the determination of FOM in serum or plasma [10, 12, 14, 15, 20, 22] 325
and urine [11]. However, these methods often require a significant amount of time 326
in sample preparation or technique, and none offer a chromatographic system 327
suitable for a pharmacokinetic study of FOM in both plasma and urine. 328
3.3. Pharmacokinetic analysis 329
The plasma concentration-time profile obtained in the pilot pharmacokinetic study is 330
shown in Figure 4. The peak plasma concentration in this patient after receiving the 331
initial dose was 222 µg/mL, and the trough concentration was 141 µg/mL. Increased 332
plasma concentrations were observed after receiving the fifth dose of FOM, with the 333
peak plasma concentration recorded as 868 µg/mL, and the trough concentration 334
was 592 µg/mL. The urinary concentration determined from a 6 h sample taken 335
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post-dose and was 2.03 mg/mL after the initial dose and 0.29 mg/mL after the fifth 336
dose of FOM. 337
4. Conclusion 338
The developed analytical method is a sensitive, simple and robust tool to analyse 339
FOM in plasma and urine of patients. With the increasing prevalence of MDR 340
organisms and the reduced effectiveness of currently available antibiotics this 341
method allows the opportunity to study the disposition of FOM, particularly in at-risk 342
patient groups. This research may improve dosing strategies which could minimize 343
the risk of increasing resistance and bring an effective antibiotic back into the hands 344
of treating physicians. 345
346
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347
Table 1. MS conditions for FOM and EPA 348
MS FOM EPA
Product Ion 137.1 (M.H-) 109.1 (M.H-)
Daughter Ion 78.9 78.9
Dwell Time (ms) 100 100
Q1 (V) +14 +10
Q3 (V) +28 +13
Collision Energy (V) +25 +20
349
Table 2: Lower limit of quantification 350
Matrix Mean Precision (%) Accuracy (%)
Plasma (n = 13) 1.02 µg/mL ±4.7 +1.7
Urine (n = 7) 0.10 mg/mL ±3.1 +1.2
351
Table 3: Linearity analysis 352
Matrix Calibration Range Correlation Coefficient
(Mean)
Maximum deviation*
(%)
Plasma (n=12) 1 to 2000 µg/mL 0.9963 -14.0
Urine (n=5) 0.1 to 10 mg/mL 0.9959 +10.5
* Reported maximum deviation from nominal (%) across all standard curves and all concentration levels. 353
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354
Table 4: Inter-assay Precision and Accuracy 355
Matrix Concentration Replicates Mean Accuracy (%) Precision (%)
Pla
sma
3 μg /mL 22 2.93 -2.4 9.1
80 μg /mL 27 78.0 -3.1 7.8
800 μg/mL 22 826 3.3 4.3
1600 μg/mL 23 1486 -7.2 5.9
Uri
ne
0.3 mg /mL 9 0.30 0.0 4.2
2 mg /mL 9 2.0 1.6 8.1
8 mg/mL 9 7.9 -1.9 2.3
356
357
Table 5: Matrix, recovery and freeze-thaw stability studies 358
359
Study Matrix Concentration Mean Accuracy (%) Precision (%)
Mat
rix
Plasma 3 μg /mL 1.05a 8.8
800 μg /mL 0.98a 3.9
Urine 0.2 mg/mL 0.189 4.6 7.2
5 mg/mL 5.36 -5.4 7.1
Rec
ove
ry
Plasma 80 μg /mL 68 7.7
Urine 10 μg/mL 98 4.2
Stab
ility
(fr
eeze
-th
aw)
Plasma 0.3 μg /mL 0.31 3.2 4.0
5 μg /mL 4.8 -3.1 5.4
80 μg /mL 86 7.2 5.8
Urine 0.3 mg /mL 0.30 0.9 2.7
2.1 mg /mL 2.3 8.2 3.1
7.8 mg /mL 8.2 4.4 1.0 a matrix factor: calculated as a ratio of peak area of FOM in the presence of matrix to the peak area in 360
the absence of matrix (normalized using the internal standard). 361
362
363
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364
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462
463
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Figure 1: Structure of fosfomycin (FOM, left) and the internal standard,
ethylphosphonic acid (EPA, right),[29,30].
Figure 1
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Figure 2: Chromatograms of a blank sample (top) and the LLOQ (1 µg/mL) plasma
standard (FOM, centre; EPA, bottom).
1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25
0
10
20
30
40137.10>78.90(-)
1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25
0
500
1000
1500
2000
137.10>78.90(-)
FFM
1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25
0
100000
200000
300000
400000
500000
109.10>78.90(-)
EPA
Time (min)
Intensity
(cps)
Figure 2
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Figure 3: Chromatograms of blank sample (top) and the LLOQ (0.1 mg/mL) urine
standard (FOM, centre; EPA, bottom)
0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
0
25
50
75
100137.10>78.90(-)
0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
0
1000
2000
3000
4000
5000
6000
137.10>78.90(-)
FFM
0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
0
25000
50000
75000
100000109.10>78.90(-)
EPA
Time (min)
Intensity
(cps)
Figure 3
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Figure 4: Plasma concentration – time profiles of FOM in a critically ill patient
receiving a 6 g FOM IV dose every 6 hours, for the first and fifth doses.
0
200
400
600
800
1000
0 1 2 3 4
FOM plasma concentration
(μg/mL )
Time (h)
Dose 1
Dose 5
Figure 4